Phosphorothioate-conjugated miRNAs and methods of using the same

ABSTRACT

Provided herein are, inter alia, nucleic acid conjugates including a non-cell penetrating ribonucleic acid compound attached at its 3′ end to a phosphorothioate polymer. Attachment of the phosphorothioate polymer to the non-cell penetrating ribonucleic acid conveys stability to and allows for efficient intracellular delivery of the non-cell penetrating ribonucleic acid. The nucleic acid conjugates provided herein including embodiments thereof are useful, inter alia, for the treatment of cancer, inflammatory disease, and pain.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/430,327, filed Jun. 3, 2019, issued as U.S. Pat. No. 10,987,428,which claims priority to U.S. Provisional Application No. 62/679,596,filed Jun. 1, 2018, which is hereby incorporated by reference in itsentirety and for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made using support under Grant Number CA122976awarded by the National Institutes of Health. The government has certainrights to this invention.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED AS AN ASCII FILE

The Sequence Listing written in file 048440-685001US_SL_ST25, created onJun. 3, 2019, 8,799 bytes, machine format IBM-PC, MS Windows operatingsystem, is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

The manipulation of intracellular molecules involved in diseasepathology is an attractive treatment option for numerous diseases. Theability to modify the activity of intracellular molecules can haveprofound effects on intracellular signaling pathways and result inchanges in gene expression that promote favorable disease outcome.However, targeting intracellular molecules for therapeutic purposes isparticularly challenging due to the need for the targeting molecule toboth penetrate the cell membrane and maintain biostability in aphysiological environment. Thus, there is a need in the art forbiostable, cell-penetrating compositions capable of targetingintracellular molecules involved in disease pathology. Provided hereinare, inter alia, compositions and methods addressing these and otherneeds in the art.

BRIEF SUMMARY OF THE INVENTION

In an aspect is provided a nucleic acid conjugate including: (i) anon-cell penetrating ribonucleic acid compound including the sequence ofSEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5; (ii)a phosphorothioate polymer; and (iii) a chemical linker attaching thephosphorothioate polymer to the 3′ end of the non-cell penetratingribonucleic acid compound; wherein the phosphorothioate polymer enhancesintracellular delivery of the non-cell penetrating nucleic acidcompound.

In an aspect is provided a nucleic acid conjugate including: (i) anon-cell penetrating ribonucleic acid compound including the sequence ofSEQ ID NO: 1; (ii) a phosphorothioate polymer; and (iii) a chemicallinker attaching the phosphorothioate polymer to the 3′ end of thenon-cell penetrating ribonucleic acid compound; wherein thephosphorothioate polymer enhances intracellular delivery of the non-cellpenetrating nucleic acid compound.

In an aspect is provided a nucleic acid conjugate including: (i) anon-cell penetrating ribonucleic acid compound including the sequence ofSEQ ID NO:2; (ii) a phosphorothioate polymer; and (iii) a chemicallinker attaching the phosphorothioate polymer to the 3′ end of thenon-cell penetrating ribonucleic acid compound; wherein thephosphorothioate polymer enhances intracellular delivery of the non-cellpenetrating nucleic acid compound.

In an aspect is provided a nucleic acid conjugate including: (i) anon-cell penetrating ribonucleic acid compound including the sequence ofSEQ ID NO:3; (ii) a phosphorothioate polymer; and (iii) a chemicallinker attaching the phosphorothioate polymer to the 3′ end of thenon-cell penetrating ribonucleic acid compound; wherein thephosphorothioate polymer enhances intracellular delivery of the non-cellpenetrating nucleic acid compound.

In an aspect is provided a nucleic acid conjugate including: (i) anon-cell penetrating ribonucleic acid compound including the sequence ofSEQ ID NO:4; (ii) a phosphorothioate polymer; and (iii) a chemicallinker attaching the phosphorothioate polymer to the 3′ end of thenon-cell penetrating ribonucleic acid compound; wherein thephosphorothioate polymer enhances intracellular delivery of the non-cellpenetrating nucleic acid compound.

In an aspect is provided a nucleic acid conjugate including: (i) anon-cell penetrating ribonucleic acid compound including the sequence ofSEQ ID NO:5; (ii) a phosphorothioate polymer; and (iii) a chemicallinker attaching the phosphorothioate polymer to the 3′ end of thenon-cell penetrating ribonucleic acid compound; wherein thephosphorothioate polymer enhances intracellular delivery of the non-cellpenetrating nucleic acid compound.

In an aspect is provided a cell including the nucleic acid conjugateprovided herein including embodiments thereof.

In an aspect is provided a pharmaceutical composition including thenucleic acid conjugate as provided herein including embodiments thereofand a pharmaceutically acceptable carrier.

In an aspect is provided a method of treating cancer in a subject inneed thereof. The method includes administering to the subject atherapeutically effective amount of a cell penetrating nucleic acidconjugate as provided herein including embodiments thereof, therebytreating the cancer in the subject.

In an aspect is provided a method of increasing expression of p53 in acancer cell, the method including contacting a cancer cell with aneffective amount of a cell penetrating nucleic acid conjugate asprovided herein including embodiments thereof, thereby increasingexpression of p53 in the cancer cell.

In an aspect is provided a method of inhibiting tumor vascularization ina subject in need thereof, the method including administering to thesubject a therapeutically effective amount of a cell penetrating nucleicacid conjugate as provided herein including embodiments thereof, therebyinhibiting tumor vascularization in the subject.

In an aspect is provided a method of treating an inflammatory disease ina subject in need thereof, the method including administering to thesubject a therapeutically effective amount of a cell penetrating nucleicacid conjugate as provided herein including embodiments thereof, therebytreating an inflammatory disease in the subject.

In an aspect is provided a method of treating pain in a subject in needthereof, the method including administering to the subject atherapeutically effective amount of a cell penetrating nucleic acidconjugate as provided herein including embodiments thereof, therebytreating pain in the subject.

In an aspect is provided a method of inhibiting IL-6 signaling in acell, the method including contacting a cell with an effective amount ofa cell penetrating nucleic acid conjugate as provided herein includingembodiments thereof, thereby inhibiting IL-6 signaling in the cell.

In an aspect is provided a method of delivering a non-cell penetratingnucleic acid into a cell, the method including contacting a cell withthe cell penetrating nucleic acid conjugate as provided herein includingembodiments thereof, thereby delivering the non-cell penetrating nucleicacid into the cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 : Modified let7a-3p miRNA (SEQ ID NO: 1). A nucleic acidconjugate including a stretch of non-cell penetrating let7a-3p miRNA, aphosphorothioated nucleic acid, and a chemical linker attaching thephosphorothioated nucleic acid to the let7a-3p miRNA, is shown. Toenhance intracellular delivery, the non-cell penetrating let7a-3p miRNAsequence was extended at its 3′-end by a chemical linker followed by a20meric ssDNA stretch. To achieve cellular internalization, it iscritical that the sugar-phosphate backbone is phosphorothioated (PS;bottom sequence of FIG. 1 ) indicated by (*); a control miRNA was usedwithout phosphorothioation (phosphodiester, PO; top sequence of FIG. 1).

FIGS. 2A-2C: Intracellular delivery of let7a-3p miRNA (SEQ ID NO: 1) andreduced STAT3 target gene expression. FIG. 2A Human multiple myelomacells MM.1S were incubated for 30 min with 10 μg/ml modified let7a-3pmiRNA as indicated and analyzed by flow cytometry. FIG. 2B. Humanmultiple myeloma cells MM.1S were incubated for 48 hrs with 10 μg/mlmodified let7a-3p miRNA 48 hrs and analyzed by flow cytometry; left peakis blank control, middle peak is let7a-3p-PO; right peak is let7a-3p-PS.FIG. 2C. Human multiple myeloma cells MM.1S were incubated for 48 hrsdaily with 10 μg/ml modified let7a-3p miRNA as indicated and analyzed byRT-PCR to assess expression of the STAT3 target oncogenic Bcl-xL gene.PS means that the indicated let7a-3p miRNA was elongated withphosphorothioate nucleic acids while PO means that the let7a-3p miRNAwas elongated with non-phosphorothioated nucleic acids. SD shown;T-test: **) P<0.01.

FIG. 3 : Modified let7a-5p miRNA (SEQ ID NO:2). A nucleic acid conjugatecomprising a stretch of non-cell penetrating let7a-5p miRNA, aphosphorothioated nucleic acid, and a chemical linker attaching thephosphorothioated nucleic acid to the let7a-5p miRNA, is shown. Toenhance intracellular delivery, the non-cell penetrating let7a-5p miRNAsequence was extended at its 3′-end by a chemical linker followed by a20meric ssDNA stretch. It is critical that the sugar-phosphate backboneis phosphorothioated (PS; bottom sequence of FIG. 3 ) indicated by (*)to achieve cellular internalization; a control miRNA was used withoutphosphorothioation (phosphodiester, PO; top sequence of FIG. 3 ).

FIGS. 4A-4C: Intracellular delivery of let7a-5p miRNA (SEQ ID NO:2) andreduced STAT3 target gene expression. FIG. 4A Human multiple myelomacells MM.1S were incubated for 30 min as indicated with 10 μg/mlmodified let7a-5p miRNA analyzed by flow cytometry. FIG. 4B Humanmultiple myeloma cells MM.1S were incubated for 48 hrs with 10 μg/mlmodified let7a-5p miRNA and analyzed by flow cytometry; left peak isblank control, middle peak is let7a-5p-PO; right peak is let7a-5p-PS.FIG. 4C. Human multiple myeloma cells MM.1S were incubated for 48 hrsdaily with 10 μg/ml modified let7a-5p miRNA as indicated and analyzed byRT-PCR to assess expression of the STAT3 target oncogenic Bcl-xL andIL-6 genes. PS means that the indicated let7a-5p miRNA was elongatedwith phosphorothioate nucleic acids while PO means that the let7a-5pmiRNA was elongated with non-phosphorothioated nucleic acids. SD shown;T-test: **) P<0.01.

FIG. 5 : Modified miR17-3p miRNA (SEQ ID NO:3). A nucleic acid conjugatecomprising a stretch of non-cell penetrating miR17-3p miRNA, aphosphorothioated nucleic acid, and a chemical linker attaching thephosphorothioated nucleic acid to the miR17-3p miRNA, is shown. Toenhance intracellular delivery, the miR17-3p miRNA was extended at its3′-end by a chemical linker followed by a 20meric ssDNA stretch. It iscritical that the sugar-phosphate backbone is phosphorothioated (PS;bottom sequence of FIG. 5 ) indicated by (*) to achieve cellularinternalization; a control miRNA was used without phosphorothioation(phosphodiester, PO; top sequence of FIG. 5 ).

FIGS. 6A-6C: miR17-3p miRNA (SEQ ID NO:3) intracellular delivery andreduced STAT3 target gene expression. FIG. 6A. Human multiple myelomacells MM.1S were incubated for 30 min with 20 μg/ml modified miR17-3pmiRNA as indicated and analyzed by flow cytometry. FIG. 6B. Humanmultiple myeloma cells MM.1S were incubated for 48 hrs with 10 μg/mlmodified miR17-3p miRNA and analyzed by flow cytometry; left peak isblank control, middle peak is miR17-3p-PO, and right peak ismiR17-3p-PS. FIG. 6C. Human multiple myeloma cells MM.1S were incubatedfor 48 hrs daily with 10 μg/ml modified miR17-3p miRNA as indicated andanalyzed by RT-PCR to assess expression of the STAT3 target oncogenicIL-6 gene. PS means that the indicated miR17-3p miRNA was elongated withphosphorothioate nucleic acids while PO means that the miR17-3p miRNAwas elongated with non-phosphorothioated nucleic acids. SD shown.

FIG. 7 : Modified miR17-5p miRNA (SEQ ID NO:4). A nucleic acid conjugatecomprising a stretch of non-cell penetrating miR17-5p miRNA, aphosphorothioated nucleic acid, and a chemical linker attaching thephosphorothioated nucleic acid to the miR17-5p miRNA, is shown. Toenhance intracellular delivery, the miR17-5p miRNA was extended at its3′-end by a chemical linker followed by a 20meric ssDNA stretch. It iscritical that the sugar-phosphate backbone is phosphorothioated (PS;bottom sequence of FIG. 7 ) indicated by (*) to achieve cellularinternalization; a control miRNA was used without phosphorothioation(phosphodiester, PO; top sequence of FIG. 7 ).

FIGS. 8A-8C: miR17-5p miRNA (SEQ ID NO:4) intracellular delivery andreduced STAT3 target gene expression. FIG. 8A. Human multiple myelomacells MM.1S were incubated for 30 min with 20 μg/ml modified miR17-5pmiRNA as indicated and analyzed by flow cytometry. FIG. 8B. Humanmultiple myeloma cells MM.1S were incubated for 48 hrs with 10 μg/mlmodified miR17-5p miRNA and analyzed by flow cytometry; left peak isblank control, middle peak is miR17-5p-PO, and right peak ismiR17-5p-PS. FIG. 8C. Human multiple myeloma cells MM.1S were incubatedfor 48 hrs daily with 10 μg/ml modified miR17-5p miRNA as indicated andanalyzed by RT-PCR to assess expression of the STAT3 target oncogenicBcl-xL and IL-6 genes. PS means that the indicated miR17-5p miRNA waselongated with phosphorothioate nucleic acids while PO means that themiR17-5p miRNA was elongated with non-phosphorothioated nucleic acids.SD shown.

FIG. 9 : Modified miR218-5p miRNA (SEQ ID NO:5). A nucleic acidconjugate comprising a stretch of non-cell penetrating miR218-5p miRNA,a phosphorothioated nucleic acid, and a chemical linker attaching thephosphorothioated nucleic acid to the miR218-5p miRNA, is shown. Toenhance intracellular delivery, the miR218-5p miRNA was extended at its3′-end by a chemical linker followed by a 20meric ssDNA stretch. It iscritical that the sugar-phosphate backbone is phosphorothioated (PS;bottom sequence of FIG. 9 ) indicated by (*) to achieve cellularinternalization; a control miRNA was used without phosphorothioation(phosphodiester, PO; top sequence of FIG. 9 ).

FIGS. 10A-10C: miR218-5p miRNA (SEQ ID NO:5) intracellular delivery andreduced STAT3 target gene expression. FIG. 10A. Human multiple myelomacells MM.1S were incubated for 30 min with 20 μg/ml modified miR218-5pmiRNA as indicated and analyzed by flow cytometry. FIG. 10B. Humanmultiple myeloma cells MM.1S were incubated for 48 hrs with 10 μg/mlmodified miR218-5p miRNA and analyzed by flow cytometry; left peak isblank control, middle peak is miR218-5p-PO, and right peak ismiR218-5p-PS. FIG. 10C. Human multiple myeloma cells MM.1S wereincubated for 48 hrs daily with 10 μg/ml modified miR218-5p miRNA asindicated and analyzed by RT-PCR to assess expression of the STAT3target oncogenic Bcl-xL gene. PS means that the indicated miR218-5pmiRNA was elongated with phosphorothioate nucleic acids while PO meansthat the miR218-5p miRNA was elongated with non-phosphorothioatednucleic acids. SD shown.

FIG. 11A-11B: Polymer-modified let7a-5p miRNA (SEQ ID NO:2). FIG. 11A. Anucleic acid conjugate comprising a stretch of non-cell penetratinglet7a-5p miRNA is shown. To enhance intracellular delivery, the let7a-5pmiRNA was extended at its 3′-end by a chemical linker followed by a20meric single-stranded abasic sugar-phosphate backbone polymers(referred to as “D”). It is critical that the sugar-phosphate backboneis phosphorothioated (PS; bottom sequence of FIG. 11 ) indicated by (*)to achieve cellular internalization; a control miRNA was used withoutphosphorothioation (phosphodiester, PO; top sequence of FIG. 11 ). FIG.11B. Shown is the abasic sugar-phosphate module lacking a base (anucleic acid) in comparison to basic “spacers.”

FIGS. 12A-12B: Polymer-modified let7a-5p miRNA (SEQ ID NO:2)intracellular delivery and reduced STAT3 target gene expression. FIG.12A. Human multiple myeloma cells MM.1S were incubated for 30 min with20 μg/ml polymer-modified let7a-5p miRNA as indicated and analyzed byflow cytometry. FIG. 12B. Human multiple myeloma cells MM.1S wereincubated for 48 hrs daily with 10 μg/ml polymer-modified let7a-5p miRNAas indicated and analyzed by RT-PCR to assess expression of the STAT3target oncogenic Bcl-xL gene. PS means that the indicated let7a-5p miRNAwas elongated with a phosphorothioate polymer while PO means that thelet7a-5p miRNA was elongated with a non-phosphorothioated polymer. SDshown.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

While various embodiments and aspects of the present invention are shownand described herein, it will be obvious to those skilled in the artthat such embodiments and aspects are provided by way of example only.Numerous variations, changes, and substitutions will now occur to thoseskilled in the art without departing from the invention. It should beunderstood that various alternatives to the embodiments of the inventiondescribed herein may be employed in practicing the invention.

All documents, or portions of documents, cited in the applicationincluding, without limitation, patents, patent applications, articles,books, manuals, and treatises are hereby expressly incorporated byreference in their entirety for any purpose.

The abbreviations used herein have their conventional meaning within thechemical and biological arts. The chemical structures and formulae setforth herein are constructed according to the standard rules of chemicalvalency known in the chemical arts.

As used herein, the term “about” means a range of values including thespecified value, which a person of ordinary skill in the art wouldconsider reasonably similar to the specified value. In embodiments, theterm “about” means within a standard deviation using measurementsgenerally acceptable in the art. In embodiments, about means a rangeextending to +/−10% of the specified value. In embodiments, about meansthe specified value.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight (i.e., unbranched) or branchedcarbon chain (or carbon), or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include di- and multivalentradicals, having the number of carbon atoms designated (i.e., C₁-C₁₀means one to ten carbons). Alkyl is not cyclized. Examples of saturatedhydrocarbon radicals include, but are not limited to, groups such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,sec-butyl, (cyclohexyl)methyl, homologs and isomers of, for example,n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkylgroup is one having one or more double bonds or triple bonds (e.g.alkene, alkyne). Examples of unsaturated alkyl groups include, but arenot limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl,2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and3-propynyl, 3-butynyl, and the higher homologs and isomers. An alkoxy isan alkyl attached to the remainder of the molecule via an oxygen linker(—O—).

The term “alkylene,” by itself or as part of another substituent, means,unless otherwise stated, a divalent radical derived from an alkyl, asexemplified, but not limited by, —CH₂CH₂CH₂CH₂—. Typically, an alkyl (oralkylene) group will have from 1 to 24 carbon atoms. A “lower alkyl” or“lower alkylene” is a shorter chain alkyl or alkylene group, generallyhaving eight or fewer carbon atoms. The term “alkenylene,” by itself oras part of another substituent, means, unless otherwise stated, adivalent radical derived from an alkene.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcombinations thereof, including at least one carbon atom and at leastone heteroatom selected from the group consisting of O, N, P, Si, and S,and wherein the nitrogen and sulfur atoms may optionally be oxidized,and the nitrogen heteroatom may optionally be quaternized. Heteroalkylis not cyclized. The heteroatom(s) O, N, P, S, and Si may be placed atany interior position of the heteroalkyl group or at the position atwhich the alkyl group is attached to the remainder of the molecule.Examples include, but are not limited to: —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CHO—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,—CH═CH—N(CH₃)—CH₃, —O—CH₃, —O—CH₂—CH₃, and —CN. Up to two or threeheteroatoms may be consecutive, such as, for example, —CH₂—NH—OCH₃ and—CH₂—O—Si(CH₃)₃.

Similarly, the term “heteroalkylene,” by itself or as part of anothersubstituent, means, unless otherwise stated, a divalent radical derivedfrom heteroalkyl, as exemplified, but not limited by,—CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylenegroups, heteroatoms can also occupy either or both of the chain termini(e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, andthe like). Still further, for alkylene and heteroalkylene linkinggroups, no orientation of the linking group is implied by the directionin which the formula of the linking group is written. For example, theformula —C(O)₂R′— represents both —C(O)₂R′— and —R′C(O)₂—. As describedabove, heteroalkyl groups, as used herein, include those groups that areattached to the remainder of the molecule through a heteroatom, such as—C(O)R′, —C(O)NR′, —NR′R″, —OR′, —SR′, and/or —SO₂R′. Where“heteroalkyl” is recited, followed by recitations of specificheteroalkyl groups, such as —NR′R″ or the like, it will be understoodthat the terms heteroalkyl and —NR′R″ are not redundant or mutuallyexclusive. Rather, the specific heteroalkyl groups are recited to addclarity. Thus, the term “heteroalkyl” should not be interpreted hereinas excluding specific heteroalkyl groups, such as —NR′R″ or the like.

The terms “cycloalkyl” and “heterocycloalkyl,” by themselves or incombination with other terms, mean, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl,” respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Cycloalkyl andheterocycloalkyl are non-aromatic. Examples of cycloalkyl include, butare not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like. A “cycloalkylene” and a“heterocycloalkylene,” alone or as part of another substituent, means adivalent radical derived from a cycloalkyl and heterocycloalkyl,respectively.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” includes, but is not limited to, fluoromethyl,difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl,3-bromopropyl, and the like.

The term “acyl” means, unless otherwise stated, —C(O)R where R is asubstituted or unsubstituted alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent, which can be a single ring ormultiple rings (preferably from 1 to 3 rings) that are fused together(i.e., a fused ring aryl) or linked covalently. A fused ring aryl refersto multiple rings fused together wherein at least one of the fused ringsis an aryl ring. The term “heteroaryl” refers to aryl groups (or rings)that contain at least one heteroatom such as N, O, or S, wherein thenitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. Thus, the term “heteroaryl” includesfused ring heteroaryl groups (i.e., multiple rings fused togetherwherein at least one of the fused rings is a heteroaromatic ring). A5,6-fused ring heteroarylene refers to two rings fused together, whereinone ring has 5 members and the other ring has 6 members, and wherein atleast one ring is a heteroaryl ring. Likewise, a 6,6-fused ringheteroarylene refers to two rings fused together, wherein one ring has 6members and the other ring has 6 members, and wherein at least one ringis a heteroaryl ring. And a 6,5-fused ring heteroarylene refers to tworings fused together, wherein one ring has 6 members and the other ringhas 5 members, and wherein at least one ring is a heteroaryl ring. Aheteroaryl group can be attached to the remainder of the moleculethrough a carbon or heteroatom. Non-limiting examples of aryl andheteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl,1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl,4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl,5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl,4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl,2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl,5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl,5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and6-quinolyl. Substituents for each of the above noted aryl and heteroarylring systems are selected from the group of acceptable substituentsdescribed below. An “arylene” and a “heteroarylene,” alone or as part ofanother substituent, mean a divalent radical derived from an aryl andheteroaryl, respectively. Non-limiting examples of heteroaryl groupsinclude pyridinyl, pyrimidinyl, thiophenyl, thienyl, furanyl, indolyl,benzoxadiazolyl, benzodioxolyl, benzodioxanyl, thianaphthanyl,pyrrolopyridinyl, indazolyl, quinolinyl, quinoxalinyl, pyridopyrazinyl,quinazolinonyl, benzoisoxazolyl, imidazopyridinyl, benzofuranyl,benzothienyl, benzothiophenyl, phenyl, naphthyl, biphenyl, pyrrolyl,pyrazolyl, imidazolyl, pyrazinyl, oxazolyl, isoxazolyl, thiazolyl,furylthienyl, pyridyl, pyrimidyl, benzothiazolyl, purinyl,benzimidazolyl, isoquinolyl, thiadiazolyl, oxadiazolyl, pyrrolyl,diazolyl, triazolyl, tetrazolyl, benzothiadiazolyl, isothiazolyl,pyrazolopyrimidinyl, pyrrolopyrimidinyl, benzotriazolyl, benzoxazolyl,or quinolyl. The examples above may be substituted or unsubstituted anddivalent radicals of each heteroaryl example above are non-limitingexamples of heteroarylene.

A fused ring heterocycloalkyl-aryl is an aryl fused to aheterocycloalkyl. A fused ring heterocycloalkyl-heteroaryl is aheteroaryl fused to a heterocycloalkyl. A fused ringheterocycloalkyl-cycloalkyl is a heterocycloalkyl fused to a cycloalkyl.A fused ring heterocycloalkyl-heterocycloalkyl is a heterocycloalkylfused to another heterocycloalkyl. Fused ring heterocycloalkyl-aryl,fused ring heterocycloalkyl-heteroaryl, fused ringheterocycloalkyl-cycloalkyl, or fused ringheterocycloalkyl-heterocycloalkyl may each independently beunsubstituted or substituted with one or more of the substituentsdescribed herein.

In some embodiments, each substituted or unsubstituted alkyl is asubstituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₃-C₇ cycloalkyl, each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7membered heterocycloalkyl, each substituted or unsubstituted aryl is asubstituted or unsubstituted C₆-C₁₀ aryl, and/or each substituted orunsubstituted heteroaryl is a substituted or unsubstituted 5 to 9membered heteroaryl. In some embodiments, each substituted orunsubstituted alkylene is a substituted or unsubstituted C₁-C₈ alkylene,each substituted or unsubstituted heteroalkylene is a substituted orunsubstituted 2 to 8 membered heteroalkylene, each substituted orunsubstituted cycloalkylene is a substituted or unsubstituted C₃-C₇cycloalkylene, each substituted or unsubstituted heterocycloalkylene isa substituted or unsubstituted 3 to 7 membered heterocycloalkylene, eachsubstituted or unsubstituted arylene is a substituted or unsubstitutedC₆-C₁₀ arylene, and/or each substituted or unsubstituted heteroaryleneis a substituted or unsubstituted 5 to 9 membered heteroarylene. In someembodiments, the compound is a chemical species set forth in theExamples section or Drawings.

The term “oxo,” as used herein, means an oxygen that is double bonded toa carbon atom.

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl,” and“heteroaryl”) includes both substituted and unsubstituted forms of theindicated radical. Preferred substituents for each type of radical areprovided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —NR′NR″R′″,—ONR′R″, —NR′C(O)NR″NR′″R″″, —CN, —NO₂, monophosphate (or derivativesthereof), diphosphate (or derivatives thereof), triphosphate (orderivatives thereof), in a number ranging from zero to (2m′+1), where m′is the total number of carbon atoms in such radical. R, R′, R″, R′″, andR″″ each preferably independently refer to hydrogen, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl (e.g., aryl substituted with 1-3 halogens),substituted or unsubstituted heteroaryl, substituted or unsubstitutedalkyl, alkoxy, or thioalkoxy groups, or arylalkyl groups. When acompound of the invention includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″,and R″″ group when more than one of these groups is present. When R′ andR″ are attached to the same nitrogen atom, they can be combined with thenitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example,—NR′R″ includes, but is not limited to, 1-pyrrolidinyl and4-morpholinyl. From the above discussion of substituents, one of skillin the art will understand that the term “alkyl” is meant to includegroups including carbon atoms bound to groups other than hydrogengroups, such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g.,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are varied and areselected from, for example: —OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″,—OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′,—NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″,—S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —NR′NR″R′″, —ONR′R″,—NR′C(O)NR″NR′″R″″, —CN, —NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy,and fluoro(C₁-C₄)alkyl, monophosphate (or derivatives thereof),diphosphate (or derivatives thereof), triphosphate (or derivativesthereof), in a number ranging from zero to the total number of openvalences on the aromatic ring system; and where R′, R″, R′″, and R″″ arepreferably independently selected from hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl. When a compound of the invention includes morethan one R group, for example, each of the R groups is independentlyselected as are each R′, R″, R′″, and R″″ groups when more than one ofthese groups is present.

Two or more substituents may optionally be joined to form aryl,heteroaryl, cycloalkyl, or heterocycloalkyl groups. Such so-calledring-forming substituents are typically, though not necessarily, foundattached to a cyclic base structure.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally form a ring of the formula -T-C(O)—(CRR′)_(q)—U—, whereinT and U are independently —NR—, —O—, —CRR′—, or a single bond, and q isan integer of from 0 to 3. Alternatively, two of the substituents onadjacent atoms of the aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula -A-(CH₂)_(r)—B—, wherein A and B areindependently —CRR′—, —O—, —NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′—, or asingle bond, and r is an integer of from 1 to 4. One of the single bondsof the new ring so formed may optionally be replaced with a double bond.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula —(CRR′)_(s)—X′—(C″R″R′″)_(d)—, where s and d are independentlyintegers of from 0 to 3, and X′ is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or—S(O)₂NR′—. The substituents R, R′, R″, and R′″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl.

As used herein, the terms “heteroatom” or “ring heteroatom” are meant toinclude, oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), andsilicon (Si).

A “substituent group,” as used herein, means a group selected from thefollowing moieties:

-   -   (A) oxo, halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂,        —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,        —NHC(O) NH₂, —NHSO₂H, —NHC═(O)H, —NHC(O)—OH, —NHOH, —OCF₃,        —OCHF₂, —NHSO₂CH₃, —N₃, unsubstituted alkyl, unsubstituted        heteroalkyl, unsubstituted cycloalkyl, unsubstituted        heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl,        monophosphate (or derivatives thereof), diphosphate (or        derivatives thereof), or triphosphate (or derivatives thereof),        and    -   (B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,        heteroaryl, monophosphate (or derivatives thereof), diphosphate        (or derivatives thereof), or triphosphate (or derivatives        thereof), substituted with at least one substituent selected        from:        -   (i) oxo, halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂,            —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,            —NHC(O)NHNH₂, —NHC(O) NH₂, —NHSO₂H, —NHC═(O)H, —NHC(O)—OH,            —NHOH, —OCF₃, —OCHF₂, —NHSO₂CH₃, —N₃, unsubstituted alkyl,            unsubstituted heteroalkyl, unsubstituted cycloalkyl,            unsubstituted heterocycloalkyl, unsubstituted aryl,            unsubstituted heteroaryl, monophosphate (or derivatives            thereof), diphosphate (or derivatives thereof), or            triphosphate (or derivatives thereof), and        -   (ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,            heteroaryl, monophosphate (or derivatives thereof),            diphosphate (or derivatives thereof), or triphosphate (or            derivatives thereof), substituted with at least one            substituent selected from:            -   (a) oxo, halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂,                —NO₂, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,                —NHC(O)NHNH₂, —NHC(O) NH₂, —NHSO₂H, —NHC═(O)H,                —NHC(O)—OH, —NHOH, —OCF₃, —OCHF₂, —NHSO₂CH₃, —N₃,                unsubstituted alkyl, unsubstituted heteroalkyl,                unsubstituted cycloalkyl, unsubstituted                heterocycloalkyl, unsubstituted aryl, unsubstituted                heteroaryl, monophosphate (or derivatives thereof),                diphosphate (or derivatives thereof), or triphosphate                (or derivatives thereof), and            -   (b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,                aryl, heteroaryl, monophosphate (or derivatives                thereof), diphosphate (or derivatives thereof), or                triphosphate (or derivatives thereof), substituted with                at least one substituent selected from: oxo, halogen,                —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₂Cl,                —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,                —NHC(O) NH₂, —NHSO₂H, —NHC═(O)H, —NHC(O)—OH, —NHOH,                —OCF₃, —OCHF₂, —NHSO₂CH₃, —N₃, unsubstituted alkyl,                unsubstituted heteroalkyl, unsubstituted cycloalkyl,                unsubstituted heterocycloalkyl, unsubstituted aryl,                unsubstituted heteroaryl, monophosphate (or derivatives                thereof), diphosphate (or derivatives thereof), and                triphosphate (or derivatives thereof).

A “size-limited substituent” or “size-limited substituent group,” asused herein, means a group selected from all of the substituentsdescribed above for a “substituent group,” wherein each substituted orunsubstituted alkyl is a substituted or unsubstituted C₁-C₂₀ alkyl, eachsubstituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₈cycloalkyl, each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 3 to 8 membered heterocycloalkyl, eachsubstituted or unsubstituted aryl is a substituted or unsubstitutedC₆-C₁₀ aryl, and each substituted or unsubstituted heteroaryl is asubstituted or unsubstituted 5 to 10 membered heteroaryl.

A “lower substituent” or “lower substituent group,” as used herein,means a group selected from all of the substituents described above fora “substituent group,” wherein each substituted or unsubstituted alkylis a substituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₃-C₇ cycloalkyl, each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7membered heterocycloalkyl, each substituted or unsubstituted aryl is asubstituted or unsubstituted C₆-C₁₀ aryl, and each substituted orunsubstituted heteroaryl is a substituted or unsubstituted 5 to 9membered heteroaryl.

As used herein, the term “isomers” refers to compounds having the samenumber and kind of atoms, and hence the same molecular weight, butdiffering in respect to the structural arrangement or configuration ofthe atoms.

The term “tautomer,” as used herein, refers to one of two or morestructural isomers which exist in equilibrium and which are readilyconverted from one isomeric form to another.

It will be apparent to one skilled in the art that certain compounds ofthis invention may exist in tautomeric forms, all such tautomeric formsof the compounds being within the scope of the invention.

Unless otherwise stated, structures depicted herein are also meant toinclude all stereochemical forms of the structure; i.e., the R and Sconfigurations for each asymmetric center. Therefore, singlestereochemical isomers as well as enantiomeric and diastereomericmixtures of the present compounds are within the scope of the invention.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds which differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of a hydrogen by a deuterium ortritium, or the replacement of a carbon by ¹³C- or ¹⁴C-enriched carbonare within the scope of this invention.

The symbol “

” denotes the point of attachment of a chemical moiety to the remainderof a molecule or chemical formula.

A “chemical linker” or “linker” as provided herein is a covalent linker,a non-covalent linker, a peptide linker (a linker including a peptidemoiety), a cleavable peptide linker, a substituted or unsubstitutedalkylene, substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene or substitutedor unsubstituted heteroarylene or any combination thereof. Thus, achemical linker as provided herein may include a plurality of chemicalmoieties, wherein each of the plurality of moieties can be chemicallydifferent.

The terms “a” or “an,” as used in herein means one or more. In addition,the phrase “substituted with a[n],” as used herein, means the specifiedgroup may be substituted with one or more of any or all of the namedsubstituents. For example, where a group, such as an alkyl or heteroarylgroup, is “substituted with an unsubstituted C₁-C₂₀ alkyl, orunsubstituted 2 to 20 membered heteroalkyl,” the group may contain oneor more unsubstituted C₁-C₂₀ alkyls, and/or one or more unsubstituted 2to 20 membered heteroalkyls. Moreover, where a moiety is substitutedwith an R substituent, the group may be referred to as “R-substituted.”Where a moiety is R-substituted, the moiety is substituted with at leastone R substituent and each R substituent is optionally different. Wherea particular R group is present in the description of a chemical genus,a Roman alphabetic symbol may be used to distinguish each appearance ofthat particular R group. For example, where multiple R¹ substituents arepresent, each R¹ substituent may be distinguished as R^(1A), R^(1B),R^(1C), R^(1D) etc., wherein each of R^(1A), R^(1B), R^(1C), R^(1D),etc. is defined within the scope of the definition of R¹ and optionallydifferently.

Descriptions of compounds of the present invention are limited byprinciples of chemical bonding known to those skilled in the art.Accordingly, where a group may be substituted by one or more of a numberof substituents, such substitutions are selected so as to comply withprinciples of chemical bonding and to give compounds which are notinherently unstable and/or would be known to one of ordinary skill inthe art as likely to be unstable under ambient conditions, such asaqueous, neutral, and several known physiological conditions. Forexample, a heterocycloalkyl or heteroaryl is attached to the remainderof the molecule via a ring heteroatom in compliance with principles ofchemical bonding known to those skilled in the art thereby avoidinginherently unstable compounds.

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by a person of ordinaryskill in the art. See, e.g., Singleton et al., DICTIONARY OFMICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York,NY 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, ColdSprings Harbor Press (Cold Springs Harbor, NY 1989). Any methods,devices and materials similar or equivalent to those described hereincan be used in the practice of this invention. The following definitionsare provided to facilitate understanding of certain terms usedfrequently herein and are not meant to limit the scope of the presentdisclosure.

“Nucleic acid” refers to nucleotides (e.g., deoxyribonucleotides orribonucleotides) and polymers thereof in either single-, double- ormultiple-stranded form, or complements thereof. The terms“polynucleotide,” “oligonucleotide,” “oligo” or the like refer, in theusual and customary sense, to a linear sequence of nucleotides. The term“nucleotide” refers, in the usual and customary sense, to a single unitof a polynucleotide, i.e., a monomer. Nucleotides can beribonucleotides, deoxyribonucleotides, or modified versions thereof.Examples of polynucleotides contemplated herein include single anddouble stranded DNA, single and double stranded RNA, and hybridmolecules having mixtures of single and double stranded DNA and RNA.Examples of nucleic acid, e.g. polynucleotides contemplated hereininclude any types of RNA, e.g. mRNA, siRNA, miRNA, and guide RNA and anytypes of DNA, genomic DNA, plasmid DNA, and minicircle DNA, and anyfragments thereof. The term “duplex” in the context of polynucleotidesrefers, in the usual and customary sense, to double strandedness.Nucleic acids can be linear or branched. For example, nucleic acids canbe a linear chain of nucleotides or the nucleic acids can be branched,e.g., such that the nucleic acids comprise one or more arms or branchesof nucleotides. Optionally, the branched nucleic acids are repetitivelybranched to form higher ordered structures such as dendrimers and thelike.

Nucleic acids, including e.g., nucleic acids with a phosphothioatebackbone, can include one or more reactive moieties. As used herein, theterm reactive moiety includes any group capable of reacting with anothermolecule, e.g., a nucleic acid or polypeptide through covalent,non-covalent or other interactions. By way of example, the nucleic acidcan include an amino acid reactive moiety that reacts with an amino acidon a protein or polypeptide through a covalent, non-covalent or otherinteraction.

The terms also encompass nucleic acids containing known nucleotideanalogs or modified backbone residues or linkages, which are synthetic,naturally occurring, and non-naturally occurring, which have similarbinding properties as the reference nucleic acid, and which aremetabolized in a manner similar to the reference nucleotides. Examplesof such analogs include, include, without limitation, phosphodiesterderivatives including, e.g., phosphoramidate, phosphorodiamidate,phosphorothioate (also known as phosphothioate having double bondedsulfur replacing oxygen in the phosphate), phosphorodithioate,phosphonocarboxylic acids, phosphonocarboxylates, phosphonoacetic acid,phosphonoformic acid, methyl phosphonate, boron phosphonate, orO-methylphosphoroamidite linkages (see Eckstein, OLIGONUCLEOTIDES ANDANALOGUES: A PRACTICAL APPROACH, Oxford University Press) as well asmodifications to the nucleotide bases such as in 5-methyl cytidine orpseudouridine; and peptide nucleic acid backbones and linkages. Otheranalog nucleic acids include those with positive backbones; non-ionicbackbones, modified sugars, and non-ribose backbones (e.g.phosphorodiamidate morpholino oligos, methyl phosphonates, chiral-methylphosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs)or locked nucleic acids (LNA) as known in the art), including thosedescribed in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and7, ASC Symposium Series 580, CARBOHYDRATE MODIFICATIONS IN ANTISENSERESEARCH, Sanghui & Cook, eds. Nucleic acids containing one or morecarbocyclic sugars are also included within one definition of nucleicacids. Modifications of the ribose-phosphate backbone may be done for avariety of reasons, e.g., to increase the stability and half-life ofsuch molecules in physiological environments or as probes on a biochip.Mixtures of naturally occurring nucleic acids and analogs can be made;alternatively, mixtures of different nucleic acid analogs, and mixturesof naturally occurring nucleic acids and analogs may be made. Inembodiments, the internucleotide linkages in DNA are phosphodiester,phosphodiester derivatives, or a combination of both.

Nucleic acids can include nonspecific sequences. As used herein, theterm “nonspecific sequence” refers to a nucleic acid sequence thatcontains a series of residues that are not designed to be complementaryto or are only partially complementary to any other nucleic acidsequence. By way of example, a nonspecific nucleic acid sequence is asequence of nucleic acid residues that does not function as aninhibitory nucleic acid when contacted with a cell or organism.

An “antisense nucleic acid” as referred to herein is a nucleic acid(e.g., DNA or RNA molecule) that is complementary to at least a portionof a specific target nucleic acid (e.g., STAT3) and is capable ofreducing transcription of the target nucleic acid (e.g. mRNA from DNA),reducing the translation of the target nucleic acid (e.g. mRNA),altering transcript splicing (e.g. single stranded morpholino oligo), orinterfering with the endogenous activity of the target nucleic acid.See, e.g., Weintraub, Scientific American, 262:40 (1990). Typically,synthetic antisense nucleic acids (e.g. oligonucleotides) are generallybetween 15 and 25 bases in length. Thus, antisense nucleic acids arecapable of hybridizing to (e.g. selectively hybridizing to) a targetnucleic acid (e.g., STAT3). In embodiments, the antisense nucleic acidhybridizes to the target nucleic acid (e.g., STAT3) in vitro. Inembodiments, the antisense nucleic acid hybridizes to the target nucleicacid (e.g., STAT3) in a cell. In embodiments, the antisense nucleic acidhybridizes to the target nucleic acid (e.g., STAT3) in an organism. Inembodiments, the antisense nucleic acid hybridizes to the target nucleicacid (e.g., STAT3) under physiological conditions. Antisense nucleicacids may comprise naturally occurring nucleotides or modifiednucleotides such as, e.g., phosphorothioate, methylphosphonate, and-anomeric sugar-phosphate, backbone modified nucleotides.

In the cell, the antisense nucleic acids hybridize to the correspondingRNA (e.g., STAT3) forming a double-stranded molecule. The antisensenucleic acids interfere with the endogenous behavior of the RNA (e.g.,STAT3) and inhibit its function relative to the absence of the antisensenucleic acid. Furthermore, the double-stranded molecule may be degradedvia the RNAi pathway. The use of antisense methods to inhibit the invitro translation of genes is well known in the art (Marcus-Sakura,Anal. Biochem., 172:289, (1988)). Further, antisense molecules whichbind directly to the DNA may be used. Antisense nucleic acids may besingle or double stranded nucleic acids. Non-limiting examples ofantisense nucleic acids include siRNAs (including their derivatives orpre-cursors, such as nucleotide analogs), short hairpin RNAs (shRNA),micro RNAs (miRNA), saRNAs (small activating RNAs) and small nucleolarRNAs (snoRNA) or certain of their derivatives or pre-cursors.

MicroRNAs (miRNAs) are a conserved class of small non-coding RNAs.miRNAs function as post-transcriptional regulators of gene expression,which are integral to almost all known biological processes, includingcell growth, proliferation and differentiation as well as organismalmetabolism and development. In the rapidly growing field of miRNAs, manymiRNAs have been identified to be functionally associated with promotingeither disease progression or cancer cell differentiation favoring animproved therapeutic prognosis. However, while miRNA sequences are easyto produce, a “gymnotic” administration of man-made unmodified RNAsequences fails to provide the desired therapeutic benefit.

The term “polymeric” refers to a molecule including repeating subunits(e.g., polymerized monomers). For example, polymeric molecules may bebased upon polyethylene glycol (PEG), poly[amino(1-oxo-1,6-hexanediyl)],poly(oxy-1,2-ethanediyloxycarbonyl-1,4-phenylenecarbonyl), tetraethyleneglycol (TEG), polyvinylpyrrolidone (PVP), poly(xylene), orpoly(p-xylylene). See, for example, “Chemistry of Protein Conjugationand Cross-Linking” Shan S. Wong CRC Press, Boca Raton, Fla., USA, 1993;“BioConjugate Techniques” Greg T. Hermanson Academic Press, San Diego,Calif., USA, 1996; “Catalog of Polyethylene Glycol and Derivatives forAdvanced PEGylation, 2004” Nektar Therapeutics Inc, Huntsville, Ala.,USA, which are incorporated by reference in their entirety for allpurposes.

The term “polymerizable monomer” is used in accordance with its meaningin the art of polymer chemistry and refers to a compound that maycovalently bind chemically to other monomer molecules (such as otherpolymerizable monomers that are the same or different) to form apolymer.

The term “block copolymer” is used in accordance with its ordinarymeaning and refers to two or more portions (e.g., blocks) of polymerizedmonomers linked by a covalent bond. In embodiments, a block copolymer isa repeating pattern of polymers. In embodiments, the block copolymerincludes two or more monomers in a periodic (e.g., repeating pattern)sequence. For example, a diblock copolymer has the formula:-B-B-B-B-B-B-A-A-A-A-A-, where ‘B’ is a first subunit and ‘A’ is asecond subunit covalently bound together. A triblock copolymer thereforeis a copolymer with three distinct blocks, two of which may be the same(e.g., -A-A-A-A-A-B-B-B-B-B-B-A-A-A-A-A-) or all three are different(e.g., -A-A-A-A-A-B-B-B-B-B-B-C-C-C-C-C-) where ‘A’ is a first subunit,‘B’ is a second subunit, and ‘C’ is a third subunit, covalently boundtogether.

The term “amphiphilic polymer” as used herein refers to a polymercontaining both hydrophilic and hydrophobic portions. In embodiments,the hydrophilic to hydrophobic portions are present in a 1 to 1 massratio. In embodiments, the hydrophilic to hydrophobic portions arepresent in a 1 to 2 mass ratio. In embodiments, the hydrophilic tohydrophobic portions are present in a 1 to 5 mass ratio. In embodiments,the hydrophilic to hydrophobic portions are present in a 2 to 1 massratio. In embodiments, the hydrophilic to hydrophobic portions arepresent in a 5 to 1 mass ratio. An amphiphilic polymer may be a diblockor triblock copolymer. In embodiments, the amphiphilic polymer mayinclude two hydrophilic portions (e.g., blocks) and one hydrophobicportion (e.g., block). In embodiments, the hydrophilic block tohydrophobic to hydrophilic ratio is 1 to 1 to 1. In embodiments, thehydrophilic block to hydrophobic to hydrophilic ratio is 1.8 to 1 to1.8. In embodiments, the hydrophilic block to hydrophobic to hydrophilicratio is 2 to 1 to 2. In embodiments, the hydrophilic block tohydrophobic to hydrophilic ratio is 1 to 1 to 2.

The term “complement,” as used herein, refers to a nucleotide (e.g., RNAor DNA) or a sequence of nucleotides capable of base pairing with acomplementary nucleotide or sequence of nucleotides. As described hereinand commonly known in the art the complementary (matching) nucleotide ofadenosine is thymidine and the complementary (matching) nucleotide ofguanidine is cytosine. Thus, a complement may include a sequence ofnucleotides that base pair with corresponding complementary nucleotidesof a second nucleic acid sequence. The nucleotides of a complement maypartially or completely match the nucleotides of the second nucleic acidsequence. Where the nucleotides of the complement completely match eachnucleotide of the second nucleic acid sequence, the complement formsbase pairs with each nucleotide of the second nucleic acid sequence.Where the nucleotides of the complement partially match the nucleotidesof the second nucleic acid sequence only some of the nucleotides of thecomplement form base pairs with nucleotides of the second nucleic acidsequence. Examples of complementary sequences include coding and anon-coding sequences, wherein the non-coding sequence containscomplementary nucleotides to the coding sequence and thus forms thecomplement of the coding sequence. A further example of complementarysequences are sense and antisense sequences, wherein the sense sequencecontains complementary nucleotides to the antisense sequence and thusforms the complement of the antisense sequence.

As described herein the complementarity of sequences may be partial, inwhich only some of the nucleic acids match according to base pairing, orcomplete, where all the nucleic acids match according to base pairing.Thus, two sequences that are complementary to each other, may have aspecified percentage of nucleotides that are the same (i.e., about 60%identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or higher identity over a specified region).

A polynucleotide is typically composed of a specific sequence of fournucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine(T) (uracil (U) for thymine (T) when the polynucleotide is RNA). Thus,the term “polynucleotide sequence” is the alphabetical representation ofa polynucleotide molecule; alternatively, the term may be applied to thepolynucleotide molecule itself. This alphabetical representation can beinput into databases in a computer having a central processing unit andused for bioinformatics applications such as functional genomics andhomology searching. Polynucleotides may optionally include one or morenon-standard nucleotide(s), nucleotide analog(s) and/or modifiednucleotides.

“Percentage of sequence identity” is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide or polypeptide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. The percentage is calculatedby determining the number of positions at which the identical nucleicacid base or amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison andmultiplying the result by 100 to yield the percentage of sequenceidentity.

An amino acid or nucleotide base “position” is denoted by a number thatsequentially identifies each amino acid (or nucleotide base) in thereference sequence based on its position relative to the N-terminus (or5′-end). Due to deletions, insertions, truncations, fusions, and thelike that must be taken into account when determining an optimalalignment, in general the amino acid residue number in a test sequencedetermined by simply counting from the N-terminus will not necessarilybe the same as the number of its corresponding position in the referencesequence. For example, in a case where a variant has a deletion relativeto an aligned reference sequence, there will be no amino acid in thevariant that corresponds to a position in the reference sequence at thesite of deletion. Where there is an insertion in an aligned referencesequence, that insertion will not correspond to a numbered amino acidposition in the reference sequence. In the case of truncations orfusions there can be stretches of amino acids in either the reference oraligned sequence that do not correspond to any amino acid in thecorresponding sequence.

The term “phosphorothioate nucleic acid” refers to a nucleic acid inwhich one or more internucleotide linkages are through aphosphorothioate moiety (thiophosphate) moiety. The phosphorothioatemoiety may be a monothiophosphate (—P(O)₃(S)³⁻—) or a dithiophosphate(—P(O)₂(S)₂ ³⁻—). In embodiments of all the aspects provided herein, thephosphorothioate moiety is a monothiophosphate (—P(O)₃(S)³⁻—). That is,in embodiments of all the aspects provided herein, the phosphorothioatenucleic acid is a monothiophosphate nucleic acid. In embodiments, one ormore of the nucleosides of a phosphorothioate nucleic acid are linkedthrough a phosphorothioate moiety (e.g. monothiophosphate) moiety, andthe remaining nucleosides are linked through a phosphodiester moiety(—P(O)₄ ³⁻—). In embodiments, one or more of the nucleosides of aphosphorothioate nucleic acid are linked through a phosphorothioatemoiety (e.g. monothiophosphate) moiety, and the remaining nucleosidesare linked through a methylphosphonate linkage. In embodiments, all thenucleosides of a phosphorothioate nucleic acid are linked through aphosphorothioate moiety (e.g. a monothiophosphate) moiety.

Phosphorothioate oligonucleotides (phosphorothioate nucleic acids) aretypically from about 5, 6, 7, 8, 9, 10, 12, 15, 25, 30, 40, 50 or morenucleotides in length, up to about 100 nucleotides in length.Phosphorothioate nucleic acids may also be longer in lengths, e.g., 200,300, 500, 1000, 2000, 3000, 5000, 7000, 10,000, etc. As described above,in certain embodiments. the phosphorothioate nucleic acids hereincontain one or more phosphodiester bonds. In other embodiments, thephosphorothioate nucleic acids include alternate backbones (e.g., mimicsor analogs of phosphodiesters as known in the art, such as,boranophosphate, methylphosphonate, phosphoramidate, orO-methylphosphoroamidite linkages (see Eckstein, Oligonucleotides andAnalogues: A Practical Approach, Oxford University Press). Thephosphorothioate nucleic acids may also include one or more nucleic acidanalog monomers known in the art, such as, peptide nucleic acid monomeror polymer, locked nucleic acid monomer or polymer, morpholino monomeror polymer, glycol nucleic acid monomer or polymer, or threose nucleicacid monomer or polymer. Other analog nucleic acids include those withpositive backbones; non-ionic backbones, and nonribose backbones,including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, andChapters 6 and 7, ASC Symposium Series 580, Carbohydrate Modificationsin Antisense Research, Sanghui & Cook, eds. Nucleic acids containing oneor more carbocyclic sugars are also included within one definition ofnucleic acids. Modifications of the ribose-phosphate backbone may bedone for a variety of reasons, e.g., to increase the stability andhalf-life of such molecules in physiological environments or as probeson a biochip. Mixtures of naturally occurring nucleic acids and analogscan be made; alternatively, mixtures of different nucleic acid analogs,and mixtures of naturally occurring nucleic acids and analogs may bemade. Phosphorothioate nucleic acids and phosphorothioate polymerbackbones can be linear or branched. For example, the branched nucleicacids are repetitively branched to form higher ordered structures suchas dendrimers and the like.

As used herein, a “phosphorothioate polymer backbone” is a chemicalpolymer with at least two phosphorothioate linkages (e.g.monothiophosphate) (e.g. linking together sugar subunits, cyclicsubunits or alkyl subunits). The phosphorothioate polymer backbone maybe a phosphorothioate sugar polymer (i.e., a polymer composed of abasicsugar-phosphate modules), which is a phosphorothioate nucleic acid inwhich one or more (or all) of the chain of pentose sugars lack the bases(nucleobases) normally present in a nucleic acid. The phosphorothioatepolymer backbone can include two or more phosphorothioate linkages. Thephosphorothioate polymer backbone can include 5, 6, 7, 8, 9, 10, 12, 15,25, 30, 40, 50 or more linkages and can contain up to about 100phosphorothioate linkages. Phosphorothioate polymer backbones may alsocontain a larger number of linkages, e.g., 200, 300, 500, 1000, 2000,3000, 5000, 7000, 10,000, and the like.

The phosphorothioate nucleic acids and phosphorothioate polymerbackbones may be partially or completely phosphorothioated. For example,50% or more of the internucleotide linkages of a phosphorothioatenucleic acid can be phosphorothioate linkages. Optionally, 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 99% of the internucleotide linkages of a phosphorothioatenucleic acid are phosphorothioate linkages. Optionally, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the internucleotidelinkages of a phosphorothioate nucleic acid are phosphorothioatelinkages. Optionally, 75%, 80%, 85%, 90%, 95%, or 99% of theinternucleotide linkages of a phosphorothioate nucleic acid arephosphorothioate linkages. Optionally, 90%, 95%, or 99% of theinternucleotide linkages of a phosphorothioate nucleic acid arephosphorothioate linkages. In embodiments, the remaining internucleotidelinkages are phosphodiester linkages. In embodiments, the remaininginternucleotide linkages are methylphosphonate linkages. Optionally,100% of the internucleotide linkages of the phosphorothioate nucleicacids are phosphorothioate linkages. Similarly, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or99%, of the intersugar linkages in a phosphorothioate polymer backbonecan be phosphorothioate linkages. Optionally, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or 99%, of the intersugar linkages in aphosphorothioate polymer backbone can be phosphorothioate linkages.Optionally, 75%, 80%, 85%, 90%, 95%, or 99%, of the intersugar linkagesin a phosphorothioate polymer backbone can be phosphorothioate linkages.Optionally, 90%, 95%, or 99%, of the intersugar linkages in aphosphorothioate polymer backbone can be phosphorothioate linkages. Inembodiments, the remaining internucleotide linkages are phosphodiesterlinkages. In embodiments, the remaining internucleotide linkages aremethylphosphonate linkages. Optionally, 100% of the intersugar linkagesof the phosphorothioate polymer backbone are phosphorothioate linkages.

A “labeled nucleic acid or oligonucleotide” is one that is bound, eithercovalently, through a linker or a chemical bond, or noncovalently,through ionic, van der Waals, electrostatic, or hydrogen bonds to alabel such that the presence of the nucleic acid may be detected bydetecting the presence of the detectable label bound to the nucleicacid. Alternatively, a method using high affinity interactions mayachieve the same results where one of a pair of binding partners bindsto the other, e.g., biotin, streptavidin. In embodiments, thephosphorothioate nucleic acid or phosphorothioate polymer backboneincludes a detectable label, as disclosed herein and generally known inthe art. In embodiments, the phosphorothioate nucleic acid orphosphorothioate polymer backbone is connected to a detectable labelthrough a chemical linker.

A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, chemical, orother physical means. For example, useful labels include 32P,fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonlyused in an ELISA), biotin, digoxigenin, or haptens and proteins or otherentities which can be made detectable, e.g., by incorporating aradiolabel into a peptide or antibody specifically reactive with atarget peptide. Any appropriate method known in the art for conjugatingan antibody to the label may be employed, e.g., using methods describedin Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., SanDiego.

The phosphorothioate nucleic acids and phosphorothioate polymerbackbones provided herein can include one or more reactive moieties,e.g., a covalent reactive moiety. A reactive moiety may be attached tothe remainder of the phosphorothioate nucleic acids and phosphorothioatepolymer backbones using any appropriate linker, such as a polymer linkerknown in the art or alternatively a polyethylene glycol linker orequivalent. The linker may, in embodiments, include (i.e. be attachedto) a detectable label as described herein. As used herein, the term“covalent reactive moiety” refers to a chemical moiety capable ofchemically reactive with an amino acid of a non-cell penetratingprotein, as described herein, to form a covalent bond and, thus, aconjugate as provided herein.

The term “gene” means the segment of DNA involved in producing aprotein; it includes regions preceding and following the coding region(leader and trailer) as well as intervening sequences (introns) betweenindividual coding segments (exons). The leader, the trailer as well asthe introns include regulatory elements that are necessary during thetranscription and the translation of a gene. Further, a “protein geneproduct” is a protein expressed from a particular gene.

The word “expression” or “expressed” as used herein in reference to agene means the transcriptional and/or translational product of thatgene. The level of expression of a DNA molecule in a cell may bedetermined on the basis of either the amount of corresponding mRNA thatis present within the cell or the amount of protein encoded by that DNAproduced by the cell.

The term “isolated”, when applied to a nucleic acid or protein, denotesthat the nucleic acid or protein is essentially free of other cellularcomponents with which it is associated in the natural state. It can be,for example, in a homogeneous state and may be in either a dry oraqueous solution. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A proteinthat is the predominant species present in a preparation issubstantially purified.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and 0-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, “conservatively modified variants” refers to those nucleicacids that encode identical or essentially identical amino acidsequences. Because of the degeneracy of the genetic code, a number ofnucleic acid sequences will encode any given protein. For instance, thecodons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, atevery position where an alanine is specified by a codon, the codon canbe altered to any of the corresponding codons described without alteringthe encoded polypeptide. Such nucleic acid variations are “silentvariations,” which are one species of conservatively modifiedvariations. Every nucleic acid sequence herein which encodes apolypeptide also describes every possible silent variation of thenucleic acid. One of skill will recognize that each codon in a nucleicacid (except AUG, which is ordinarily the only codon for methionine, andTGG, which is ordinarily the only codon for tryptophan) can be modifiedto yield a functionally identical molecule. Accordingly, each silentvariation of a nucleic acid which encodes a polypeptide is implicit ineach described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another:

-   -   1) Alanine (A), Glycine (G);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);    -   7) Serine (S), Threonine (T); and    -   8) Cysteine (C), Methionine (M)    -   (see, e.g., Creighton, Proteins (1984)).

The terms “numbered with reference to” or “corresponding to,” when usedin the context of the numbering of a given amino acid or polynucleotidesequence, refer to the numbering of the residues of a specifiedreference sequence when the given amino acid or polynucleotide sequenceis compared to the reference sequence. An amino acid residue in aprotein “corresponds” to a given residue when it occupies the sameessential structural position within the protein as the given residue.One skilled in the art will immediately recognize the identity andlocation of residues corresponding to a specific position in a protein(e.g., STAT3) in other proteins with different numbering systems. Forexample, by performing a simple sequence alignment with a protein (e.g.,STAT3) the identity and location of residues corresponding to specificpositions of said protein are identified in other protein sequencesaligning to said protein. For example, a selected residue in a selectedprotein corresponds to lysine at position 685 when the selected residueoccupies the same essential spatial or other structural relationship asa lysine at position 685. In some embodiments, where a selected proteinis aligned for maximum homology with a protein, the position in thealigned selected protein aligning with lysine 685 is said to correspondto lysine 685. Instead of a primary sequence alignment, a threedimensional structural alignment can also be used, e.g., where thestructure of the selected protein is aligned for maximum correspondencewith the lysine at position 685, and the overall structures compared. Inthis case, an amino acid that occupies the same essential position aslysine 685 in the structural model is said to correspond to the lysine685 residue.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over aspecified region, when compared and aligned for maximum correspondenceover a comparison window or designated region) as measured using a BLASTor BLAST 2.0 sequence comparison algorithms with default parametersdescribed below, or by manual alignment and visual inspection (see,e.g., NCBI web site http://www.ncbi.nlm.nih.gov/BLAST/ or the like).Such sequences are then said to be “substantially identical.” Thisdefinition also refers to, or may be applied to, the compliment of atest sequence. The definition also includes sequences that havedeletions and/or additions, as well as those that have substitutions. Asdescribed below, the preferred algorithms can account for gaps and thelike. Preferably, identity exists over a region that is at least about25 amino acids or nucleotides in length, or more preferably over aregion that is 50-100 amino acids or nucleotides in length.

The terms “STAT3,” “STAT3 protein,” “STAT3 peptide” as referred toherein include any of the recombinant or naturally-occurring forms ofthe Signal transducer and activator of transcription 3 (STAT3) proteinor variants or homologs thereof that maintain STAT3 protein activity(e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%activity compared to STAT3). In some aspects, the variants or homologshave at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequenceidentity across the whole sequence or a portion of the sequence (e.g. a10, 20, 50, 100, 150 or 200 continuous amino acid portion) compared to anaturally occurring STAT3 polypeptide. In embodiments, the STAT3 peptideis substantially identical to the protein identified by the UniProtreference number P40763 or a variant or homolog having substantialidentity thereto. In embodiments, the STAT3 peptide includes thesequence of SEQ ID NO:8. In embodiments, the STAT3 peptide is thesequence of SEQ ID NO:8.

The term “IL-6” or “Interleukin 6” as referred to herein includes any ofthe recombinant or naturally-occurring forms of the IL-6 protein orvariants or homologs thereof that maintain IL-6 protein activity (e.g.within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activitycompared to IL-6). IL-6 is a member of the interleukin family and actsas both a pro-inflammatory cytokine and an anti-inflammatory myokine. Insome aspects, the variants or homologs have at least 90%, 95%, 96%, 97%,98%, 99% or 100% amino acid sequence identity across the whole sequenceor a portion of the sequence (e.g. a 50, 100, 150 or 200 continuousamino acid portion) compared to a naturally occurring IL-6 polypeptide.In embodiments, the IL-6 protein is substantially identical to theprotein identified by the UniProt reference number P05231 or a variantor homolog having substantial identity thereto.

As used herein, the terms “cell-penetrating” or “cell-penetration” referto the ability of a molecule (e.g. a protein) to pass from theextracellular environment into a cell in a significant or effectiveamount. Thus, a cell-penetrating conjugate is a molecule that passesfrom the extracellular environment, through the membrane, and into acell.

As used herein, the terms “non-cell penetrating” or “non-cellpenetration” refers to the inability of a molecule to pass from theextracellular environment into a cell in a significant or effectiveamount. Thus, non-cell penetrating nucleic acids or ribonucleic acidcompounds generally are not capable of passing from the extracellularenvironment, through the cell membrane, and into a cell in order toachieve a significant biological effect on a population of cells, organor organism. The term does not exclude the possibility that one or moreof the small number of nucleic acids or ribonucleic acid compounds mayenter the cell. However, the term refers to molecules that are generallynot able to enter a cell from the extracellular environment to asignificant degree. Examples of non-cell penetrating molecules andsubstances include, but are not limited to, large molecules such as, forexample, high molecular weight proteins, nucleic acids or ribonucleicacid compounds. Nucleic acids or ribonucleic acid compounds can bedetermined to be non-cell penetrating using methods known to those ofskill in the art. By way of example, a nucleic acid or ribonucleic acidcompound can be fluorescently labeled and the ability of the nucleicacid or ribonucleic acid compound to pass from the extracellularenvironment into the cell can be determined in vitro by flow cytometricanalysis or confocal microscopy. In some embodiments, a “non-cellpenetrating nucleic acid or ribonucleic acid compound” refers to anucleic acid or ribonucleic acid compound that penetrates a cell atleast about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 10,000 or 100,000fold less than the same nucleic acid or ribonucleic acid compoundattached to a phosphorothioate nucleic acid or phosphorothioate polymerbackbone. In some embodiments, a “non-cell penetrating nucleic acid orribonucleic acid compound” refers to a nucleic acid or ribonucleic acidcompound that does not measurably penetrate a cell.

As used herein, the term “intracellular” means inside a cell. As usedherein, an “intracellular target” is a target, e.g., nucleic acid,polypeptide or other molecule (e.g., carbohydrate) that is locatedinside of a cell and is a target to which the non-cell penetratingnucleic acids or ribonucleic acid compounds provided herein bind.Binding can be direct or indirect. Optionally, the non-cell penetratingnucleic acid or ribonucleic acid compound selectively binds theintracellular target. By selectively binds, selectively binding, orspecifically binding refers to the agent (e.g., a non-cell penetratingnucleic acid or ribonucleic acid compound) binding one agent (e.g.,intracellular target) to the partial or complete exclusion of otheragents. By binding is meant a detectable binding at least about 1.5times the background of the assay method. For selective or specificbinding such a detectable binding can be detected for a given agent butnot a control agent. Alternatively, or additionally, the detection ofbinding can be determined by assaying the presence of down-streammolecules or events.

As used herein, the term “conjugate” refers to the association betweenatoms or molecules. The association can be direct or indirect. Forexample, a conjugate between a nucleic acid and a protein or nucleicacid or ribonucleic acid compound can be direct, e.g., by covalent bond,or indirect, e.g., by non-covalent bond (e.g. electrostatic interactions(e.g. ionic bond, hydrogen bond, halogen bond), van der Waalsinteractions (e.g. dipole-dipole, dipole-induced dipole, Londondispersion), ring stacking (pi effects), hydrophobic interactions andthe like). Optionally, conjugates are formed using conjugate chemistryincluding, but are not limited to nucleophilic substitutions (e.g.,reactions of amines and alcohols with acyl halides, active esters),electrophilic substitutions (e.g., enamine reactions) and additions tocarbon-carbon and carbon-heteroatom multiple bonds (e.g., Michaelreaction, Diels-Alder addition). These and other useful reactions arediscussed in, for example, March, ADVANCED ORGANIC CHEMISTRY, 3rd Ed.,John Wiley & Sons, New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES,Academic Press, San Diego, 1996; and Feeney et al., MODIFICATION OFPROTEINS; Advances in Chemistry Series, Vol. 198, American ChemicalSociety, Washington, D.C., 1982. In embodiments, the phosphorothioatenucleic acid and phosphorothioate backbone polymer are non-covalentlyattached to the nucleic acid or ribonucleic acid compound through anon-covalent chemical reaction between a component of thephosphorothioate nucleic acid and phosphorothioate backbone polymer(e.g., a monothiophosphate) and a component of the nucleic acid orribonucleic acid compound.

A “cell” as used herein, refers to a cell carrying out metabolic orother functions sufficient to preserve or replicate its genomic DNA. Acell can be identified by well-known methods in the art including, forexample, presence of an intact membrane, staining by a particular dye,ability to produce progeny or, in the case of a gamete, ability tocombine with a second gamete to produce a viable offspring. Cells mayinclude prokaryotic and eukaroytic cells. Prokaryotic cells include butare not limited to bacteria. Eukaryotic cells include but are notlimited to yeast cells and cells derived from plants and animals, forexample mammalian, insect (e.g., Spodoptera) and human cells. Cells maybe useful when they are naturally nonadherent or have been treated notto adhere to surfaces, for example by trypsinization.

The term “activating,” as used herein, refers to an nucleic acidconjugate capable of detectably increasing the expression or activity ofa given gene or protein (e.g., p53). The activating nucleic acidconjugate can increase expression or activity 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90% or more in comparison to a control in the absence ofthe activating nucleic acid. In certain instances, expression oractivity is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or higherthan the expression or activity in the absence of the activating nucleicacid conjugate.

As defined herein, the term “inhibition”, “inhibit”, “inhibiting” andthe like in reference to an nucleic acid conjugate interaction meansnegatively affecting (e.g. decreasing) the activity or function of aprotein (e.g., STAT3) relative to the activity or function of theprotein in the absence of the inhibitor. In embodiments inhibitionrefers to reduction of a disease or symptoms of disease. Thus, inembodiments, inhibition includes, at least in part, partially or totallyblocking stimulation, decreasing, preventing, or delaying activation, orinactivating, desensitizing, or down-regulating signal transduction orenzymatic activity or the amount of a protein.

“Contacting” is used in accordance with its plain ordinary meaning andrefers to the process of allowing at least two distinct species (e.g.chemical compounds including biomolecules or cells) to becomesufficiently proximal to react, interact or physically touch. It shouldbe appreciated; however, the resulting reaction product can be produceddirectly from a reaction between the added reagents or from anintermediate from one or more of the added reagents which can beproduced in the reaction mixture.

The term “contacting” may include allowing two species to react,interact, or physically touch, wherein the two species may be, forexample, a non-cell penetrating nucleic acid compound (e.g., let7a-3p(SEQ ID NO:1), let7a-5p (SEQ ID NO:2), miR17-3p (SEQ ID NO:3), miR17-5p(SEQ ID NO:4), miR218-5p (SEQ ID NO:5)) as described herein and anintracellular target (e.g., STAT3 (SEQ ID NO:8)).

“Biological sample” or “sample” refer to materials obtained from orderived from a subject or patient. A biological sample includes sectionsof tissues such as biopsy and autopsy samples, and frozen sections takenfor histological purposes. Such samples include bodily fluids such asblood and blood fractions or products (e.g., serum, plasma, platelets,red blood cells, and the like), sputum, tissue, cultured cells (e.g.,primary cultures, explants, and transformed cells) stool, urine,synovial fluid, joint tissue, synovial tissue, synoviocytes,fibroblast-like synoviocytes, macrophage-like synoviocytes, immunecells, hematopoietic cells, fibroblasts, macrophages, T cells, etc. Abiological sample is typically obtained from a eukaryotic organism, suchas a mammal such as a primate e.g., chimpanzee or human; cow; dog; cat;a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; orfish.

A “control” or “standard control” refers to a sample, measurement, orvalue that serves as a reference, usually a known reference, forcomparison to a test sample, measurement, or value. For example, a testsample can be taken from a patient suspected of having a given disease(e.g. an autoimmune disease, inflammatory autoimmune disease, cancer,infectious disease, immune disease, or other disease) and compared to aknown normal (non-diseased) individual (e.g. a standard controlsubject). A standard control can also represent an average measurementor value gathered from a population of similar individuals (e.g.standard control subjects) that do not have a given disease (i.e.standard control population), e.g., healthy individuals with a similarmedical background, same age, weight, etc. A standard control value canalso be obtained from the same individual, e.g. from an earlier-obtainedsample from the patient prior to disease onset. One of skill willrecognize that standard controls can be designed for assessment of anynumber of parameters (e.g. RNA levels, protein levels, specific celltypes, specific bodily fluids, specific tissues, synoviocytes, synovialfluid, synovial tissue, fibroblast-like synoviocytes, macrophagelikesynoviocytes, etc).

One of skill in the art will understand which standard controls are mostappropriate in a given situation and be able to analyze data based oncomparisons to standard control values. Standard controls are alsovaluable for determining the significance (e.g. statisticalsignificance) of data. For example, if values for a given parameter arewidely variant in standard controls, variation in test samples will notbe considered as significant.

The terms “subject,” “patient,” “individual,” etc. are not intended tobe limiting and can be generally interchanged. That is, an individualdescribed as a “patient” does not necessarily have a given disease, butmay be merely seeking medical advice.

The terms “disease” or “condition” refer to a state of being or healthstatus of a patient or subject capable of being treated with a compound,pharmaceutical composition, or method provided herein. In embodiments,the disease is cancer (e.g. lung cancer, ovarian cancer, osteosarcoma,bladder cancer, cervical cancer, liver cancer, kidney cancer, skincancer (e.g., Merkel cell carcinoma), testicular cancer, leukemia,lymphoma, head and neck cancer, colorectal cancer, prostate cancer,pancreatic cancer, melanoma, breast cancer, neuroblastoma). The diseasemay be an autoimmune, inflammatory, cancer, infectious, metabolic,developmental, cardiovascular, liver, intestinal, endocrine,neurological, or other disease.

As used herein, the term “cancer” refers to all types of cancer,neoplasm or malignant tumors found in mammals, including leukemias,lymphomas, melanomas, neuroendocrine tumors, carcinomas and sarcomas.Exemplary cancers that may be treated with a compound, pharmaceuticalcomposition, or method provided herein include lymphoma (cutaneousT-cell lymphoma), sarcoma, bladder cancer, bone cancer, brain tumor,cervical cancer, colon cancer, esophageal cancer, gastric cancer, headand neck cancer, kidney cancer, myeloma, thyroid cancer, leukemia,prostate cancer, breast cancer (e.g. triple negative, ER positive, ERnegative, chemotherapy resistant, herceptin resistant, HER2 positive,doxorubicin resistant, tamoxifen resistant, ductal carcinoma, lobularcarcinoma, primary, metastatic), ovarian cancer, pancreatic cancer,liver cancer (e.g., hepatocellular carcinoma), lung cancer (e.g.non-small cell lung carcinoma, squamous cell lung carcinoma,adenocarcinoma, large cell lung carcinoma, small cell lung carcinoma,carcinoid, sarcoma), glioblastoma multiforme, glioma, melanoma, prostatecancer, castration-resistant prostate cancer, breast cancer, triplenegative breast cancer, glioblastoma, ovarian cancer, lung cancer,squamous cell carcinoma (e.g., head, neck, or esophagus), colorectalcancer, leukemia, acute myeloid leukemia, lymphoma, B cell lymphoma, ormultiple myeloma. Additional examples include, cancer of the thyroid,endocrine system, brain, breast, cervix, colon, head & neck, esophagus,liver, kidney, lung, non-small cell lung, melanoma, mesothelioma, ovary,sarcoma, stomach, uterus or Medulloblastoma, Hodgkin's Disease,Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, glioma,glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primarythrombocytosis, primary macroglobulinemia, primary brain tumors, cancer,malignant pancreatic insulanoma, malignant carcinoid, urinary bladdercancer, premalignant skin lesions, testicular cancer, lymphomas, thyroidcancer, neuroblastoma, esophageal cancer, genitourinary tract cancer,malignant hypercalcemia, endometrial cancer, adrenal cortical cancer,neoplasms of the endocrine or exocrine pancreas, medullary thyroidcancer, medullary thyroid carcinoma, melanoma, colorectal cancer,papillary thyroid cancer, hepatocellular carcinoma, Paget's Disease ofthe Nipple, Phyllodes Tumors, Lobular Carcinoma, Ductal Carcinoma,cancer of the pancreatic stellate cells, cancer of the hepatic stellatecells, or prostate cancer.

The term “leukemia” refers broadly to progressive, malignant diseases ofthe blood-forming organs and is generally characterized by a distortedproliferation and development of leukocytes and their precursors in theblood and bone marrow. Leukemia is generally clinically classified onthe basis of (1) the duration and character of the disease-acute orchronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid(lymphogenous), or monocytic; and (3) the increase or non-increase inthe number abnormal cells in the blood-leukemic or aleukemic(subleukemic). Exemplary leukemias that may be treated with a compound,pharmaceutical composition, or method provided herein include, forexample, acute nonlymphocytic leukemia, chronic lymphocytic leukemia,acute granulocytic leukemia, chronic granulocytic leukemia, acutepromyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, aleukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovineleukemia, chronic myelocytic leukemia, leukemia cutis, embryonalleukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia,hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia,stem cell leukemia, acute monocytic leukemia, leukopenic leukemia,lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia,lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia,mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia,monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloidgranulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasmacell leukemia, multiple myeloma, plasmacytic leukemia, promyelocyticleukemia, Rieder cell leukemia, Schilling's leukemia, stem cellleukemia, subleukemic leukemia, or undifferentiated cell leukemia.

The term “sarcoma” generally refers to a tumor which is made up of asubstance like the embryonic connective tissue and is generally composedof closely packed cells embedded in a fibrillar or homogeneoussubstance. Sarcomas that may be treated with a compound, pharmaceuticalcomposition, or method provided herein include a chondrosarcoma,fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma,Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft partsarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma,chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrialsarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblasticsarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma,idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcomaof B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen'ssarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma,leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma,reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovialsarcoma, or telangiectaltic sarcoma.

The term “melanoma” is taken to mean a tumor arising from themelanocytic system of the skin and other organs. Melanomas that may betreated with a compound, pharmaceutical composition, or method providedherein include, for example, acral-lentiginous melanoma, amelanoticmelanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma,Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma,malignant melanoma, nodular melanoma, subungal melanoma, or superficialspreading melanoma.

The term “carcinoma” refers to a malignant new growth made up ofepithelial cells tending to infiltrate the surrounding tissues and giverise to metastases. Exemplary carcinomas that may be treated with acompound, pharmaceutical composition, or method provided herein include,for example, medullary thyroid carcinoma, familial medullary thyroidcarcinoma, acinar carcinoma, acinous carcinoma, adenocystic carcinoma,adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenalcortex, alveolar carcinoma, alveolar cell carcinoma, basal cellcarcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamouscell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma,bronchogenic carcinoma, cerebriform carcinoma, cholangiocellularcarcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma,corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinomacutaneum, cylindrical carcinoma, cylindrical cell carcinoma, ductcarcinoma, ductal carcinoma, carcinoma durum, embryonal carcinoma,encephaloid carcinoma, epiermoid carcinoma, carcinoma epithelialeadenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum,gelatiniforni carcinoma, gelatinous carcinoma, giant cell carcinoma,carcinoma gigantocellulare, glandular carcinoma, granulosa cellcarcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellularcarcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypernephroidcarcinoma, infantile embryonal carcinoma, carcinoma in situ,intraepidermal carcinoma, intraepithelial carcinoma, Krompecher'scarcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticularcarcinoma, carcinoma lenticulare, lipomatous carcinoma, lobularcarcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullarycarcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma,carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma,carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes,nasopharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans,osteoid carcinoma, papillary carcinoma, periportal carcinoma,preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma,renal cell carcinoma of kidney, reserve cell carcinoma, carcinomasarcomatodes, schneiderian carcinoma, scirrhous carcinoma, carcinomascroti, signet-ring cell carcinoma, carcinoma simplex, small-cellcarcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cellcarcinoma, carcinoma spongiosum, squamous carcinoma, squamous cellcarcinoma, string carcinoma, carcinoma telangiectaticum, carcinomatelangiectodes, transitional cell carcinoma, carcinoma tuberosum,tubular carcinoma, tuberous carcinoma, verrucous carcinoma, or carcinomavillosum.

As used herein, the terms “metastasis,” “metastatic,” and “metastaticcancer” can be used interchangeably and refer to the spread of aproliferative disease or disorder, e.g., cancer, from one organ oranother non-adjacent organ or body part. Cancer occurs at an originatingsite, e.g., breast, which site is referred to as a primary tumor, e.g.,primary breast cancer. Some cancer cells in the primary tumor ororiginating site acquire the ability to penetrate and infiltratesurrounding normal tissue in the local area and/or the ability topenetrate the walls of the lymphatic system or vascular systemcirculating through the system to other sites and tissues in the body. Asecond clinically detectable tumor formed from cancer cells of a primarytumor is referred to as a metastatic or secondary tumor. When cancercells metastasize, the metastatic tumor and its cells are presumed to besimilar to those of the original tumor. Thus, if lung cancermetastasizes to the breast, the secondary tumor at the site of thebreast consists of abnormal lung cells and not abnormal breast cells.The secondary tumor in the breast is referred to a metastatic lungcancer. Thus, the phrase metastatic cancer refers to a disease in whicha subject has or had a primary tumor and has one or more secondarytumors. The phrases non-metastatic cancer or subjects with cancer thatis not metastatic refers to diseases in which subjects have a primarytumor but not one or more secondary tumors. For example, metastatic lungcancer refers to a disease in a subject with or with a history of aprimary lung tumor and with one or more secondary tumors at a secondlocation or multiple locations, e.g., in the breast.

As used herein, “treating” or “treatment of” a condition, disease ordisorder or symptoms associated with a condition, disease or disorderrefers to an approach for obtaining beneficial or desired results,including clinical results. Beneficial or desired clinical results caninclude, but are not limited to, alleviation or amelioration of one ormore symptoms or conditions, diminishment of extent of condition,disorder or disease, stabilization of the state of condition, disorderor disease, prevention of development of condition, disorder or disease,prevention of spread of condition, disorder or disease, delay or slowingof condition, disorder or disease progression, delay or slowing ofcondition, disorder or disease onset, amelioration or palliation of thecondition, disorder or disease state, and remission, whether partial ortotal. “Treating” can also mean prolonging survival of a subject beyondthat expected in the absence of treatment. “Treating” can also meaninhibiting the progression of the condition, disorder or disease,slowing the progression of the condition, disorder or diseasetemporarily, although in some instances, it involves halting theprogression of the condition, disorder or disease permanently. As usedherein the terms treatment, treat, or treating refers to a method ofreducing the effects of one or more symptoms of a disease or conditioncharacterized by expression of the protease or symptom of the disease orcondition characterized by expression of the protease. Thus in thedisclosed method, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or 100% reduction in the severity of an establisheddisease, condition, or symptom of the disease or condition. For example,a method for treating a disease is considered to be a treatment if thereis a 10% reduction in one or more symptoms of the disease in a subjectas compared to a control. Thus the reduction can be a 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between10% and 100% as compared to native or control levels. It is understoodthat treatment does not necessarily refer to a cure or complete ablationof the disease, condition, or symptoms of the disease or condition.Further, as used herein, references to decreasing, reducing, orinhibiting include a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90% or greater as compared to a control level and such terms can includebut do not necessarily include complete elimination.

The terms “dose” and “dosage” are used interchangeably herein. A doserefers to the amount of active ingredient given to an individual at eachadministration. The dose will vary depending on a number of factors,including the range of normal doses for a given therapy, frequency ofadministration; size and tolerance of the individual; severity of thecondition; risk of side effects; and the route of administration. One ofskill will recognize that the dose can be modified depending on theabove factors or based on therapeutic progress. The term “dosage form”refers to the particular format of the pharmaceutical or pharmaceuticalcomposition, and depends on the route of administration. For example, adosage form can be in a liquid form for nebulization, e.g., forinhalants, in a tablet or liquid, e.g., for oral delivery, or a salinesolution, e.g., for injection.

As used herein, the term “administering” means oral administration,administration as a suppository, topical contact, intravenous,parenteral, intraperitoneal, intramuscular, intralesional, intrathecal,intranasal or subcutaneous administration, or the implantation of aslow-release device, e.g., a mini-osmotic pump, to a subject.Administration is by any route, including parenteral and transmucosal(e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, ortransdermal). Parenteral administration includes, e.g., intravenous,intramuscular, intra-arteriole, intradermal, subcutaneous,intraperitoneal, intraventricular, and intracranial. Other modes ofdelivery include, but are not limited to, the use of liposomalformulations, intravenous infusion, transdermal patches, etc.

By “therapeutically effective dose or amount” as used herein is meant adose that produces effects for which it is administered (e.g. treatingor preventing a disease). The exact dose and formulation will depend onthe purpose of the treatment, and will be ascertainable by one skilledin the art using known techniques (see, e.g., Lieberman, PharmaceuticalDosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technologyof Pharmaceutical Compounding (1999); Remington: The Science andPractice of Pharmacy, 20th Edition, Gennaro, Editor (2003), and Pickar,Dosage Calculations (1999)). For example, for the given parameter, atherapeutically effective amount will show an increase or decrease of atleast 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least100%. Therapeutic efficacy can also be expressed as “-fold” increase ordecrease. For example, a therapeutically effective amount can have atleast a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over astandard control. A therapeutically effective dose or amount mayameliorate one or more symptoms of a disease. A therapeuticallyeffective dose or amount may prevent or delay the onset of a disease orone or more symptoms of a disease when the effect for which it is beingadministered is to treat a person who is at risk of developing thedisease.

As used herein, the term “pharmaceutically acceptable” is usedsynonymously with “physiologically acceptable” and “pharmacologicallyacceptable”. A pharmaceutical composition will generally comprise agentsfor buffering and preservation in storage, and can include buffers andcarriers for appropriate delivery, depending on the route ofadministration.

Nucleic Acid Conjugates

Provided herein are, inter alia, nucleic acid conjugates including anon-cell penetrating ribonucleic acid compound attached at its 3′ end toa phosphorothioate polymer. The ribonucleic acid compounds conjugated tophophorothioate polymers at their 3′ end exhibit surprising biostabilityand can be delivered intracellulary with high efficiency. Upon entryinto a cell the non-cell penetrating ribonucleic acid compounds providedherein may target and modify the activity of intracellular moleculesinvolved in disease pathology thereby improving disease outcome. Thenucleic acid conjugates provided herein including embodiments thereofare useful, inter alia, for the treatment of cancer, inflammatorydisease, and pain.

In an aspect is provided a nucleic acid conjugate including: (i) anon-cell penetrating ribonucleic acid compound including the sequence ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5; (ii)a phosphorothioate polymer; and (iii) a chemical linker attaching thephosphorothioate polymer to the 3′ end of the non-cell penetratingribonucleic acid compound; wherein the phosphorothioate polymer enhancesintracellular delivery of the non-cell penetrating nucleic acidcompound.

In embodiments, the non-cell penetrating ribonucleic acid compoundincludes the sequence of SEQ ID NO: 1. In embodiments, the non-cellpenetrating ribonucleic acid compound includes the sequence of SEQ IDNO:2. In embodiments, the non-cell penetrating ribonucleic acid compoundincludes the sequence of SEQ ID NO:3. In embodiments, the non-cellpenetrating ribonucleic acid compound includes the sequence of SEQ IDNO:4. In embodiments, the non-cell penetrating ribonucleic acid compoundincludes the sequence of SEQ ID NO:5.

In embodiments, the non-cell penetrating ribonucleic acid compound isthe sequence of SEQ ID NO:1. In embodiments, the non-cell penetratingribonucleic acid compound is the sequence of SEQ ID NO:2. Inembodiments, the non-cell penetrating ribonucleic acid compound is thesequence of SEQ ID NO:3. In embodiments, the non-cell penetratingribonucleic acid compound is the sequence of SEQ ID NO:4. Inembodiments, the non-cell penetrating ribonucleic acid compound is thesequence of SEQ ID NO:5.

In embodiments, the non-cell penetrating ribonucleic acid compound is amicro RNA (miRNA).

In embodiments, the non-cell penetrating ribonucleic acid compound isabout 10, 20, 30, 40, 50, 60, 70, 80, 90 or more residues in length. Inembodiments, the non-cell penetrating ribonucleic acid compound is morethan about 10 residues in length. In embodiments, the non-cellpenetrating ribonucleic acid compound is more than about 20 residues inlength. In embodiments, the non-cell penetrating ribonucleic acidcompound is more than about 30 residues in length. In embodiments, thenon-cell penetrating ribonucleic acid compound is more than about 40residues in length. In embodiments, the non-cell penetrating ribonucleicacid compound is more than about 50 residues in length. In embodiments,the non-cell penetrating ribonucleic acid compound is more than about 60residues in length. In embodiments, the non-cell penetrating ribonucleicacid compound is more than about 70 residues in length. In embodiments,the non-cell penetrating ribonucleic acid compound is more than about 80residues in length. In embodiments, the non-cell penetrating ribonucleicacid compound is more than about 90 residues in length.

In embodiments, the non-cell penetrating ribonucleic acid compound isabout 10 residues in length. In embodiments, the non-cell penetratingribonucleic acid compound is 10 residues in length. In embodiments, thenon-cell penetrating ribonucleic acid compound is about 20 residues inlength. In embodiments, the non-cell penetrating ribonucleic acidcompound is 20 residues in length.

In embodiments, the non-cell penetrating ribonucleic acid compound isabout 21 residues in length. In embodiments, the non-cell penetratingribonucleic acid compound is 21 residues in length. In embodiments, thenon-cell penetrating ribonucleic acid compound is about 22 residues inlength. In embodiments, the non-cell penetrating ribonucleic acidcompound is 22 residues in length. In embodiments, the non-cellpenetrating ribonucleic acid compound is about 23 residues in length. Inembodiments, the non-cell penetrating ribonucleic acid compound is 23residues in length. In embodiments, the non-cell penetrating ribonucleicacid compound is about 24 residues in length. In embodiments, thenon-cell penetrating ribonucleic acid compound is 24 residues in length.In embodiments, the non-cell penetrating ribonucleic acid compound isabout 25 residues in length. In embodiments, the non-cell penetratingribonucleic acid compound is 25 residues in length. In embodiments, thenon-cell penetrating ribonucleic acid compound is about 26 residues inlength.

In embodiments, the non-cell penetrating ribonucleic acid compound is 26residues in length. In embodiments, the non-cell penetrating ribonucleicacid compound is about 27 residues in length. In embodiments, thenon-cell penetrating ribonucleic acid compound is 27 residues in length.In embodiments, the non-cell penetrating ribonucleic acid compound isabout 28 residues in length. In embodiments, the non-cell penetratingribonucleic acid compound is 28 residues in length. In embodiments, thenon-cell penetrating ribonucleic acid compound is about 29 residues inlength. In embodiments, the non-cell penetrating ribonucleic acidcompound is 29 residues in length. In embodiments, the non-cellpenetrating ribonucleic acid compound is about 30 residues in length. Inembodiments, the non-cell penetrating ribonucleic acid compound is 30residues in length.

In embodiments, the non-cell penetrating ribonucleic acid compound isabout 40 residues in length. In embodiments, the non-cell penetratingribonucleic acid compound is 40 residues in length. In embodiments, thenon-cell penetrating ribonucleic acid compound is about 50 residues inlength. In embodiments, the non-cell penetrating ribonucleic acidcompound is 50 residues in length. In embodiments, the non-cellpenetrating ribonucleic acid compound is about 60 residues in length. Inembodiments, the non-cell penetrating ribonucleic acid compound is 60residues in length. In embodiments, the non-cell penetrating ribonucleicacid compound is about 70 residues in length. In embodiments, thenon-cell penetrating ribonucleic acid compound is 70 residues in length.In embodiments, the non-cell penetrating ribonucleic acid compound isabout 80 residues in length. In embodiments, the non-cell penetratingribonucleic acid compound is 80 residues in length. In embodiments, thenon-cell penetrating ribonucleic acid compound is about 90 residues inlength. In embodiments, the non-cell penetrating ribonucleic acidcompound is 90 residues in length.

In embodiments, the non-cell penetrating ribonucleic acid compound isfrom about 20 to about 30 residues in length. In embodiments, thenon-cell penetrating ribonucleic acid compound is from 20 to 30 residuesin length. In embodiments, the non-cell penetrating ribonucleic acidcompound is from about 21 to about 30 residues in length. Inembodiments, the non-cell penetrating ribonucleic acid compound is from21 to 30 residues in length. In embodiments, the non-cell penetratingribonucleic acid compound is from about 22 to about 30 residues inlength. In embodiments, the non-cell penetrating ribonucleic acidcompound is from 22 to 30 in length. In embodiments, the non-cellpenetrating ribonucleic acid compound is from about 23 to about 30residues in length. In embodiments, the non-cell penetrating ribonucleicacid compound is from 23 to 30 residues in length. In embodiments, thenon-cell penetrating ribonucleic acid compound is from about 24 to about30 residues in length. In embodiments, the non-cell penetratingribonucleic acid compound is from 24 to 30 residues in length.

In embodiments, the non-cell penetrating ribonucleic acid compound isfrom about 25 to about 30 residues in length. In embodiments, thenon-cell penetrating ribonucleic acid compound is from 25 to 30 residuesin length. In embodiments, the non-cell penetrating ribonucleic acidcompound is from about 26 to about 30 residues in length. Inembodiments, the non-cell penetrating ribonucleic acid compound is from26 to 30 residues in length. In embodiments, the non-cell penetratingribonucleic acid compound is from about 27 to about 30 residues inlength. In embodiments, the non-cell penetrating ribonucleic acidcompound is from 27 to 30 residues in length. In embodiments, thenon-cell penetrating ribonucleic acid compound is from about 28 to about30 residues in length. In embodiments, the non-cell penetratingribonucleic acid compound is from 28 to 30 residues in length.

In embodiments, the non-cell penetrating ribonucleic acid compound isfrom about 20 to about 29 residues in length. In embodiments, thenon-cell penetrating ribonucleic acid compound is from 20 to 29 residuesin length. In embodiments, the non-cell penetrating ribonucleic acidcompound is from about 20 to about 28 residues in length. Inembodiments, the non-cell penetrating ribonucleic acid compound is from20 to 28 residues in length. In embodiments, the non-cell penetratingribonucleic acid compound is from about 20 to about 27 residues inlength. In embodiments, the non-cell penetrating ribonucleic acidcompound is from 20 to 27 residues in length. In embodiments, thenon-cell penetrating ribonucleic acid compound is from about 20 to about26 residues in length. In embodiments, the non-cell penetratingribonucleic acid compound is from 20 to 26 residues in length.

In embodiments, the non-cell penetrating ribonucleic acid compound isfrom about 20 to about 25 residues in length. In embodiments, thenon-cell penetrating ribonucleic acid compound is from 20 to 25 residuesin length. In embodiments, the non-cell penetrating ribonucleic acidcompound is from about 20 to about 24 residues in length. Inembodiments, the non-cell penetrating ribonucleic acid compound is from20 to 24 residues in length. In embodiments, the non-cell penetratingribonucleic acid compound is from about 20 to about 23 residues inlength. In embodiments, the non-cell penetrating ribonucleic acidcompound is from 20 to 23 residues in length. In embodiments, thenon-cell penetrating ribonucleic acid compound is from about 20 to about22 residues in length. In embodiments, the non-cell penetratingribonucleic acid compound is from 20 to 22 residues in length.

In embodiments, the non-cell penetrating ribonucleic acid compound isfrom about 21 to about 25 residues in length. In embodiments, thenon-cell penetrating ribonucleic acid compound is from 21 to 25 residuesin length. In embodiments, the non-cell penetrating ribonucleic acidcompound is from about 22 to about 25 residues in length. Inembodiments, the non-cell penetrating ribonucleic acid compound is from22 to 25 residues in length. In embodiments, the non-cell penetratingribonucleic acid compound is from about 23 to about 25 residues inlength. In embodiments, the non-cell penetrating ribonucleic acidcompound is from 23 to 25 residues in length.

In embodiments, the phosphorothioate polymer is a phosphorothioatenucleic acid or an abasic sugar-phosphorothioated polymer. An “abasicsugar-phosphate polymer” as provided herein refers to a polymerincluding abasic sugar moieties (i.e. a moiety including a ribose ordeoxyribose aromatic ring that does not have a base attached to it,which is not substituted with a base), wherein the abasic sugar moietiesare covalently linked to other abasic sugar moieties or to sugarmoieties substituted with a base and wherein the moieties are connectedthrough a phosphodiester bond or a phosphorothioate bond. Inembodiments, the phosphorothioate polymer is a phosphorothioate nucleicacid. In embodiments, the phosphorothioate polymer is an abasicsugar-phosphorothioated polymer. In embodiments, the phosphorothioatepolymer is a phosphorothioate deoxyribonucleic acid.

In embodiments, the phosphorothioate polymer is about 10, 20, 30, 40,50, 60, 70, 80, 90, 100 or more residues in length. In embodiments, thephosphorothioate polymer is about 10 residues in length. In embodiments,the phosphorothioate polymer is 10 residues in length. In embodiments,the phosphorothioate polymer is more than about 10 residues in length.In embodiments, the phosphorothioate polymer is more than 10 residues inlength. In embodiments, the phosphorothioate polymer is about 20residues in length. In embodiments, the phosphorothioate polymer is 20residues in length. In embodiments, the phosphorothioate polymer is morethan about 20 residues in length. In embodiments, the phosphorothioatepolymer is more than 20 residues in length. In embodiments, thephosphorothioate polymer is about 30 residues in length. In embodiments,the phosphorothioate polymer is 30 residues in length. In embodiments,the phosphorothioate polymer is more than about 30 residues in length.In embodiments, the phosphorothioate polymer is more than 30 residues inlength.

In embodiments, the phosphorothioate polymer is about 40 residues inlength. In embodiments, the phosphorothioate polymer is 40 residues inlength. In embodiments, the phosphorothioate polymer is more than about40 residues in length. In embodiments, the phosphorothioate polymer ismore than 40 residues in length. In embodiments, the phosphorothioatepolymer is about 50 residues in length. In embodiments, thephosphorothioate polymer is 50 residues in length. In embodiments, thephosphorothioate polymer is more than about 50 residues in length. Inembodiments, the phosphorothioate polymer is more than 50 residues inlength. In embodiments, the phosphorothioate polymer is about 60residues in length. In embodiments, the phosphorothioate polymer is 60residues in length. In embodiments, the phosphorothioate polymer is morethan about 60 residues in length. In embodiments, the phosphorothioatepolymer is more than 60 residues in length.

In embodiments, the phosphorothioate polymer is about 70 residues inlength. In embodiments, the phosphorothioate polymer is 70 residues inlength. In embodiments, the phosphorothioate polymer is more than about70 residues in length. In embodiments, the phosphorothioate polymer ismore than 70 residues in length. In embodiments, the phosphorothioatepolymer is about 80 residues in length. In embodiments, thephosphorothioate polymer is 80 residues in length. In embodiments, thephosphorothioate polymer is more than about 80 residues in length. Inembodiments, the phosphorothioate polymer is more than 80 residues inlength. In embodiments, the phosphorothioate polymer is about 90residues in length. In embodiments, the phosphorothioate polymer is 90residues in length. In embodiments, the phosphorothioate polymer is morethan about 90 residues in length. In embodiments, the phosphorothioatepolymer is more than 90 residues in length. In embodiments, thephosphorothioate polymer is about 100 residues in length. Inembodiments, the phosphorothioate polymer is 100 residues in length.

In embodiments, the phosphorothioate polymer is from about 10 to about30 residues in length. In embodiments, the phosphorothioate polymer isfrom 10 to 30 residues in length. In embodiments, the phosphorothioatepolymer is from about 11 to about 30 residues in length. In embodiments,the phosphorothioate polymer is from 11 to 30 residues in length. Inembodiments, the phosphorothioate polymer is from about 12 to about 30residues in length. In embodiments, the phosphorothioate polymer is from12 to 30 in length. In embodiments, the phosphorothioate polymer is fromabout 13 to about 30 residues in length. In embodiments, thephosphorothioate polymer is from 13 to 30 residues in length. Inembodiments, the phosphorothioate polymer is from about 14 to about 30residues in length. In embodiments, the phosphorothioate polymer is from14 to 30 residues in length.

In embodiments, the phosphorothioate polymer is from about 15 to about30 residues in length. In embodiments, the phosphorothioate polymer isfrom 15 to 30 residues in length. In embodiments, the phosphorothioatepolymer is from about 16 to about 30 residues in length. In embodiments,the phosphorothioate polymer is from 16 to 30 residues in length. Inembodiments, the phosphorothioate polymer is from about 17 to about 30residues in length. In embodiments, the phosphorothioate polymer is from17 to 30 residues in length. In embodiments, the phosphorothioatepolymer is from about 18 to about 30 residues in length. In embodiments,the phosphorothioate polymer is from 18 to 30 residues in length. Inembodiments, the phosphorothioate polymer is from about 19 to about 30residues in length. In embodiments, the phosphorothioate polymer is from19 to 30 residues in length.

In embodiments, the phosphorothioate polymer is from about 20 to about30 residues in length. In embodiments, the phosphorothioate polymer isfrom 20 to 30 residues in length. In embodiments, the phosphorothioatepolymer is from about 21 to about 30 residues in length. In embodiments,the phosphorothioate polymer is from 21 to 30 residues in length. Inembodiments, the phosphorothioate polymer is from about 22 to about 30residues in length. In embodiments, the phosphorothioate polymer is from22 to 30 in length. In embodiments, the phosphorothioate polymer is fromabout 23 to about 30 residues in length. In embodiments, thephosphorothioate polymer is from 23 to 30 residues in length. Inembodiments, the phosphorothioate polymer is from about 24 to about 30residues in length. In embodiments, the phosphorothioate polymer is from24 to 30 residues in length.

In embodiments, the phosphorothioate polymer is from about 25 to about30 residues in length. In embodiments, the phosphorothioate polymer isfrom 25 to 30 residues in length. In embodiments, the phosphorothioatepolymer is from about 26 to about 30 residues in length. In embodiments,the phosphorothioate polymer is from 26 to 30 residues in length. Inembodiments, the phosphorothioate polymer is from about 27 to about 30residues in length. In embodiments, the phosphorothioate polymer is from27 to 30 residues in length. In embodiments, the phosphorothioatepolymer is from about 28 to about 30 residues in length. In embodiments,the phosphorothioate polymer is from 28 to 30 residues in length.

In embodiments, the phosphorothioate polymer is from about 10 to about29 residues in length. In embodiments, the phosphorothioate polymer isfrom 10 to 29 residues in length. In embodiments, the phosphorothioatepolymer is from about 10 to about 28 residues in length. In embodiments,the phosphorothioate polymer is from 10 to 28 residues in length. Inembodiments, the phosphorothioate polymer is from about 10 to about 27residues in length. In embodiments, the phosphorothioate polymer is from10 to 27 residues in length. In embodiments, the phosphorothioatepolymer is from about 10 to about 26 residues in length. In embodiments,the phosphorothioate polymer is from 10 to 26 residues in length. Inembodiments, the phosphorothioate polymer is from about 10 to about 25residues in length. In embodiments, the phosphorothioate polymer is from10 to 25 residues in length.

In embodiments, the phosphorothioate polymer is from about 10 to about24 residues in length. In embodiments, the phosphorothioate polymer isfrom 10 to 24 residues in length. In embodiments, the phosphorothioatepolymer is from about 10 to about 23 residues in length. In embodiments,the phosphorothioate polymer is from 10 to 23 residues in length. Inembodiments, the phosphorothioate polymer is from about 10 to about 22residues in length. In embodiments, the phosphorothioate polymer is from10 to 22 residues in length. In embodiments, the phosphorothioatepolymer is from about 10 to about 21 residues in length. In embodiments,the phosphorothioate polymer is from 10 to 21 residues in length.

In embodiments, the phosphorothioate polymer is from about 10 to about20 residues in length. In embodiments, the phosphorothioate polymer isfrom 10 to 20 residues in length. In embodiments, the phosphorothioatepolymer is from about 10 to about 19 residues in length. In embodiments,the phosphorothioate polymer is from 10 to 19 residues in length. Inembodiments, the phosphorothioate polymer is from about 10 to about 18residues in length. In embodiments, the phosphorothioate polymer is from10 to 18 residues in length. In embodiments, the phosphorothioatepolymer is from about 10 to about 17 residues in length. In embodiments,the phosphorothioate polymer is from 10 to 17 residues in length. Inembodiments, the phosphorothioate polymer is from about 10 to about 16residues in length. In embodiments, the phosphorothioate polymer is from10 to 16 residues in length.

In embodiments, the phosphorothioate polymer is from about 10 to about15 residues in length. In embodiments, the phosphorothioate polymer isfrom 10 to 15 residues in length. In embodiments, the phosphorothioatepolymer is from about 10 to about 14 residues in length. In embodiments,the phosphorothioate polymer is from 10 to 14 residues in length. Inembodiments, the phosphorothioate polymer is from about 10 to about 13residues in length. In embodiments, the phosphorothioate polymer is from10 to 13 residues in length. In embodiments, the phosphorothioatepolymer is from about 10 to about 12 residues in length. In embodiments,the phosphorothioate polymer is from 10 to 12 residues in length.

In embodiments, the phosphorothioate polymer is about 20 residues inlength. In embodiments, the phosphorothioate polymer is 20 residues inlength.

In embodiments, the phosphorothioate polymer includes the sequence ofSEQ ID NO:6 or SEQ ID NO:7. In embodiments, the phosphorothioate polymerincludes the sequence of SEQ ID NO:6. In embodiments, thephosphorothioate polymer is SEQ ID NO:6. In embodiments, thephosphorothioate polymer includes the sequence of SEQ ID NO:7 Inembodiments, the phosphorothioate polymer is SEQ ID NO:7.

In embodiments, the phosphorothioate polymer is single-stranded. Inembodiments, the phosphorothioate nucleic acid is a single-strandedphosphorothioate nucleic acid. In embodiments, the abasicsugar-phosphorothioated polymer is a single-stranded abasicsugar-phosphorothioated polymer.

The conjugates provided herein including embodiments thereof may includea ribonucleic acid compound attached to a phosphorothioate polymerthrough a chemical linker. In embodiments, the chemical linker is acovalent linker.

In embodiments, the chemical linker is a covalent linker. Inembodiments, the linker includes the structure of formula:

In formula (I), R¹ is hydrogen, halogen, —CF₃, —CN, —CCl₃, —COOH,—CH₂COOH, —CONH₂, —OH, —SH, —NO₂, —NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,substituted (e.g., substituted with substituent group(s), size-limitedsubstituent group(s), or lower substituent group(s)) or unsubstitutedalkyl, substituted (e.g., substituted with substituent group(s),size-limited substituent group(s), or lower substituent group(s)) orunsubstituted heteroalkyl, substituted (e.g., substituted withsubstituent group(s), size-limited substituent group(s), or lowersubstituent group(s)) or unsubstituted cycloalkyl, substituted (e.g.,substituted with substituent group(s), size-limited substituentgroup(s), or lower substituent group(s)) or unsubstitutedheterocycloalkyl, substituted (e.g., substituted with substituentgroup(s), size-limited substituent group(s), or lower substituentgroup(s)) or unsubstituted aryl, or substituted (e.g., substituted withsubstituent group(s), size-limited substituent group(s), or lowersubstituent group(s)) or unsubstituted heteroaryl.

In embodiments, R¹ is hydrogen, halogen, —CF₃, —CN, —CCl₃, —COOH,—CH₂COOH, —CONH₂, —OH, —SH, —NO₂, —NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,unsubstituted alkyl, unsubstituted heteroalkyl, unsubstitutedcycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, orunsubstituted heteroaryl.

In embodiments, the linker is -L¹-L²-L³-L⁴-L⁵-L⁶-L⁷-. In embodiments, L¹is a bond, —NH—N═CH—, —S(O)₂—, —NR²—, —O—, —S—, —C(O)—, —C(O)NH—,—NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—, substituted (e.g., substitutedwith substituent group(s), size-limited substituent group(s), or lowersubstituent group(s)) or unsubstituted alkylene, substituted (e.g.,substituted with substituent group(s), size-limited substituentgroup(s), or lower substituent group(s)) or unsubstitutedheteroalkylene, substituted (e.g., substituted with substituentgroup(s), size-limited substituent group(s), or lower substituentgroup(s)) or unsubstituted cycloalkylene, substituted (e.g., substitutedwith substituent group(s), size-limited substituent group(s), or lowersubstituent group(s)) or unsubstituted heterocycloalkylene, substituted(e.g., substituted with substituent group(s), size-limited substituentgroup(s), or lower substituent group(s)) or unsubstituted arylene, orsubstituted (e.g., substituted with substituent group(s), size-limitedsubstituent group(s), or lower substituent group(s)) or unsubstitutedheteroarylene.

In embodiments, L² is a bond, —NH—N═CH—, —S(O)₂—, —NR²—, —O—, —S—,—C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—, substituted(e.g., substituted with substituent group(s), size-limited substituentgroup(s), or lower substituent group(s)) or unsubstituted alkylene,substituted (e.g., substituted with substituent group(s), size-limitedsubstituent group(s), or lower substituent group(s)) or unsubstitutedheteroalkylene, substituted (e.g., substituted with substituentgroup(s), size-limited substituent group(s), or lower substituentgroup(s)) or unsubstituted cycloalkylene, substituted (e.g., substitutedwith substituent group(s), size-limited substituent group(s), or lowersubstituent group(s)) or unsubstituted heterocycloalkylene, substituted(e.g., substituted with substituent group(s), size-limited substituentgroup(s), or lower substituent group(s)) or unsubstituted arylene, orsubstituted (e.g., substituted with substituent group(s), size-limitedsubstituent group(s), or lower substituent group(s)) or unsubstitutedheteroarylene.

In embodiments, L³ is a bond, —NH—N═CH—, —S(O)₂—, —NR²—, —O—, —S—,—C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—, substituted(e.g., substituted with substituent group(s), size-limited substituentgroup(s), or lower substituent group(s)) or unsubstituted alkylene,substituted (e.g., substituted with substituent group(s), size-limitedsubstituent group(s), or lower substituent group(s)) or unsubstitutedheteroalkylene, substituted (e.g., substituted with substituentgroup(s), size-limited substituent group(s), or lower substituentgroup(s)) or unsubstituted cycloalkylene, substituted (e.g., substitutedwith substituent group(s), size-limited substituent group(s), or lowersubstituent group(s)) or unsubstituted heterocycloalkylene, substituted(e.g., substituted with substituent group(s), size-limited substituentgroup(s), or lower substituent group(s)) or unsubstituted arylene, orsubstituted (e.g., substituted with substituent group(s), size-limitedsubstituent group(s), or lower substituent group(s)) or unsubstitutedheteroarylene.

In embodiments, L⁴ is a bond, —NH—N═CH—, —S(O)₂—, —NR²—, —O—, —S—,—C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—, substituted(e.g., substituted with substituent group(s), size-limited substituentgroup(s), or lower substituent group(s)) or unsubstituted alkylene,substituted (e.g., substituted with substituent group(s), size-limitedsubstituent group(s), or lower substituent group(s)) or unsubstitutedheteroalkylene, substituted (e.g., substituted with substituentgroup(s), size-limited substituent group(s), or lower substituentgroup(s)) or unsubstituted cycloalkylene, substituted (e.g., substitutedwith substituent group(s), size-limited substituent group(s), or lowersubstituent group(s)) or unsubstituted heterocycloalkylene, substituted(e.g., substituted with substituent group(s), size-limited substituentgroup(s), or lower substituent group(s)) or unsubstituted arylene, orsubstituted (e.g., substituted with substituent group(s), size-limitedsubstituent group(s), or lower substituent group(s)) or unsubstitutedheteroarylene.

In embodiments, L⁵ is a bond, —NH—N═CH—, —S(O)₂—, —NR²—, —O—, —S—,—C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—, substituted(e.g., substituted with substituent group(s), size-limited substituentgroup(s), or lower substituent group(s)) or unsubstituted alkylene,substituted (e.g., substituted with substituent group(s), size-limitedsubstituent group(s), or lower substituent group(s)) or unsubstitutedheteroalkylene, substituted (e.g., substituted with substituentgroup(s), size-limited substituent group(s), or lower substituentgroup(s)) or unsubstituted cycloalkylene, substituted (e.g., substitutedwith substituent group(s), size-limited substituent group(s), or lowersubstituent group(s)) or unsubstituted heterocycloalkylene, substituted(e.g., substituted with substituent group(s), size-limited substituentgroup(s), or lower substituent group(s)) or unsubstituted arylene, orsubstituted (e.g., substituted with substituent group(s), size-limitedsubstituent group(s), or lower substituent group(s)) or unsubstitutedheteroarylene.

In embodiments, L⁶ is a bond, —NH—N═CH—, —S(O)₂—, —NR²—, —O—, —S—,—C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—, substituted(e.g., substituted with substituent group(s), size-limited substituentgroup(s), or lower substituent group(s)) or unsubstituted alkylene,substituted (e.g., substituted with substituent group(s), size-limitedsubstituent group(s), or lower substituent group(s)) or unsubstitutedheteroalkylene, substituted (e.g., substituted with substituentgroup(s), size-limited substituent group(s), or lower substituentgroup(s)) or unsubstituted cycloalkylene, substituted (e.g., substitutedwith substituent group(s), size-limited substituent group(s), or lowersubstituent group(s)) or unsubstituted heterocycloalkylene, substituted(e.g., substituted with substituent group(s), size-limited substituentgroup(s), or lower substituent group(s)) or unsubstituted arylene, orsubstituted (e.g., substituted with substituent group(s), size-limitedsubstituent group(s), or lower substituent group(s)) or unsubstitutedheteroarylene.

In embodiments, L⁷ is a bond, —NH—N═CH—, —S(O)₂—, —NR²—, —O—, —S—,—C(O)—, —C(O)NH—, —NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—, substituted(e.g., substituted with substituent group(s), size-limited substituentgroup(s), or lower substituent group(s)) or unsubstituted alkylene,substituted (e.g., substituted with substituent group(s), size-limitedsubstituent group(s), or lower substituent group(s)) or unsubstitutedheteroalkylene, substituted (e.g., substituted with substituentgroup(s), size-limited substituent group(s), or lower substituentgroup(s)) or unsubstituted cycloalkylene, substituted (e.g., substitutedwith substituent group(s), size-limited substituent group(s), or lowersubstituent group(s)) or unsubstituted heterocycloalkylene, substituted(e.g., substituted with substituent group(s), size-limited substituentgroup(s), or lower substituent group(s)) or unsubstituted arylene, orsubstituted (e.g., substituted with substituent group(s), size-limitedsubstituent group(s), or lower substituent group(s)) or unsubstitutedheteroarylene.

In embodiments, the linker is a non-immunogenic linker.

In embodiments, the conjugate includes a detectable moiety. Inembodiments, the detectable moiety is attached to the non-cellpenetrating ribonucleic acid compound. In embodiments, the detectablemoiety is attached to the phosphorothioate polymer. In embodiments, thedetectable moiety forms part of the linker. In embodiments, thedetectable moiety is covalently attached to the linker.

The non-cell penetrating ribonucleic acid compounds provided hereinincluding embodiments thereof are useful for the treatment of cancer,inflammatory disease, and/or pain by modifying the activity ofintracellular molecules. In embodiments, the conjugate as providedherein including embodiments thereof is bound to an intracellulartarget. Thus, in embodiments, the non-cell penetrating ribonucleic acidcompound inhibits the activity or expression of an intracellular target.In embodiments, the non-cell penetrating ribonucleic acid compoundinhibits the activity of an intracellular target. In embodiments, thenon-cell penetrating ribonucleic acid compound inhibits the expressionof an intracellular target. In embodiments, the intracellular target isa signaling molecule or transcription factor. In embodiments, theintracellular target is a signaling molecule. In embodiments, theintracellular target is a transcription factor. In embodiments, thesignaling molecule is a phosphatase or kinase. In embodiments, thesignaling molecule is a phosphatase. In embodiments, the signalingmolecule is a kinase.

In embodiments, the intracellular target is a transcription factor. Inembodiments, the intracellular target is STAT3. In embodiments, theintracellular target is a STAT3 protein including the amino acidsequence of SEQ ID NO:8. In embodiments, the intracellular target is theamino acid sequence of SEQ ID NO:8. [

In embodiments, the non-cell penetrating ribonucleic acid compoundincludes a ribonucleic acid compound which inhibits STAT3 activityrelative to a standard control. In embodiments, the non-cell penetratingribonucleic acid compound is a ribonucleic acid compound which inhibitsSTAT3 activity relative to a standard control. In embodiments, thenon-cell penetrating ribonucleic acid compound includes a ribonucleicacid compound, which inhibits expression of a STAT3 target gene relativeto a standard control. In embodiments, the non-cell penetratingribonucleic acid compound is a ribonucleic acid compound, which inhibitsexpression of a STAT3 target gene relative to a standard control. Inembodiments, the STAT3 target gene is an oncogene. In embodiments, theSTAT3 target gene comprises Bcl-xL or IL-6. In embodiments, the STAT3target gene comprises Bcl-xL. In embodiments, the STAT3 target genecomprises IL-6. In embodiments, the STAT3 target gene is Bcl-xL or IL-6.In embodiments, the STAT3 target gene is Bcl-xL. In embodiments, theSTAT3 target gene is IL-6.

In an aspect is provided a cell including a nucleic acid conjugate asdescribed herein including embodiments thereof. In embodiments, the cellis a breast cancer cell, a prostate cancer cell, an ovarian cancer cell,a brain cancer cell, a pancreatic cancer cell, a melanoma cell, a coloncancer cell, a gastric cancer cell, a head-and-neck cancer cell, a livercancer cell, a lung cancer cell, a cervical cancer cell, a sarcoma cell,a leukemia cell, a lymphoma cell, a multiple myeloma cell or ametastatic lung cancer cell. In embodiments, the cell is a breast cancercell. In embodiments, the cell is a prostate cancer cell. Inembodiments, the cell is an ovarian cancer cell. In embodiments, thecell is a brain cancer cell. In embodiments, the cell is a pancreaticcancer cell. In embodiments, the cell is a melanoma cell. Inembodiments, the cell is a colon cancer cell. In embodiments, the cellis a gastric cancer cell. In embodiments, the cell is a head-and-neckcancer cell. In embodiments, the cell is a liver cancer cell. Inembodiments, the cell is a lung cancer cell. In embodiments, the cell isa cervical cancer cell. In embodiments, the cell is a sarcoma cell. Inembodiments, the cell is a leukemia cell. In embodiments, the cell is alymphoma cell. In embodiments, the cell is a multiple myeloma cell. Inembodiments, the cell is a metastatic lung cancer cell.

Pharmaceutical Compositions

The conjugates provided herein including embodiments thereof are furthercontemplated as forming part of a pharmaceutical composition. Therefore,in an aspect is provided a pharmaceutical composition including thenucleic acid conjugate as described herein including embodiments thereofand a pharmaceutically acceptable carrier.

Pharmaceutical compositions provided by the present invention includecompositions wherein the active ingredient (e.g. compositions describedherein, including embodiments thereof) is contained in a therapeuticallyeffective amount, i.e., in an amount effective to achieve its intendedpurpose. The actual amount effective for a particular application willdepend, inter alia, on the condition being treated. When administered inmethods to treat a disease, the conjugates described herein will containan amount of active ingredient effective to achieve the desired result,e.g., modulating the activity of a target molecule, and/or reducing,eliminating, or slowing the progression of disease symptoms.Determination of a therapeutically effective amount of a conjugate ofthe invention is within the capabilities of those skilled in the art.

The compositions for administration will commonly include an agent asdescribed herein dissolved in a pharmaceutically acceptable carrier,preferably an aqueous carrier. A variety of aqueous carriers can beused, e.g., buffered saline and the like. These solutions are sterileand generally free of undesirable matter. These compositions may besterilized by conventional, well known sterilization techniques. Thecompositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, toxicity adjusting agents and thelike, for example, sodium acetate, sodium chloride, potassium chloride,calcium chloride, sodium lactate and the like. The concentration ofactive agent in these formulations can vary widely, and will be selectedprimarily based on fluid volumes, viscosities, body weight and the likein accordance with the particular mode of administration selected andthe subject's needs.

Solutions of the active compounds as free base or pharmacologicallyacceptable salt can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations can contain a preservative to prevent the growth ofmicroorganisms.

Pharmaceutical compositions can be delivered via intranasal or inhalablesolutions or sprays, aerosols or inhalants. Nasal solutions can beaqueous solutions designed to be administered to the nasal passages indrops or sprays. Nasal solutions can be prepared so that they aresimilar in many respects to nasal secretions. Thus, the aqueous nasalsolutions usually are isotonic and slightly buffered to maintain a pH of5.5 to 6.5. In addition, antimicrobial preservatives, similar to thoseused in ophthalmic preparations and appropriate drug stabilizers, ifrequired, may be included in the formulation. Various commercial nasalpreparations are known and can include, for example, antibiotics andantihistamines.

Oral formulations can include excipients as, for example, pharmaceuticalgrades of mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, cellulose, magnesium carbonate and the like. Thesecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders. In someembodiments, oral pharmaceutical compositions will comprise an inertdiluent or assimilable edible carrier, or they may be enclosed in hardor soft shell gelatin capsule, or they may be compressed into tablets,or they may be incorporated directly with the food of the diet. For oraltherapeutic administration, the active compounds may be incorporatedwith excipients and used in the form of ingestible tablets, buccaltablets, troches, capsules, elixirs, suspensions, syrups, wafers, andthe like. Such compositions and preparations should contain at least0.1% of active compound. The percentage of the compositions andpreparations may, of course, be varied and may conveniently be betweenabout 2 to about 75% of the weight of the unit, or preferably between25-60%. The amount of active compounds in such compositions is such thata suitable dosage can be obtained.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered and the liquid diluent firstrendered isotonic with sufficient saline or glucose. Aqueous solutions,in particular, sterile aqueous media, are especially suitable forintravenous, intramuscular, subcutaneous and intraperitonealadministration. For example, one dosage could be dissolved in 1 ml ofisotonic NaCl solution and either added to 1000 ml of hypodermoclysisfluid or injected at the proposed site of infusion.

Sterile injectable solutions can be prepared by incorporating the activecompounds or constructs in the required amount in the appropriatesolvent followed by filtered sterilization. Generally, dispersions areprepared by incorporating the various sterilized active ingredients intoa sterile vehicle which contains the basic dispersion medium.Vacuum-drying and freeze-drying techniques, which yield a powder of theactive ingredient plus any additional desired ingredients, can be usedto prepare sterile powders for reconstitution of sterile injectablesolutions. The preparation of more, or highly, concentrated solutionsfor direct injection is also contemplated. DMSO can be used as solventfor extremely rapid penetration, delivering high concentrations of theactive agents to a small area.

The formulations of compounds can be presented in unit-dose ormulti-dose sealed containers, such as ampules and vials. Thus, thecomposition can be in unit dosage form. In such form the preparation issubdivided into unit doses containing appropriate quantities of theactive component. Thus, the compositions can be administered in avariety of unit dosage forms depending upon the method ofadministration. For example, unit dosage forms suitable for oraladministration include, but are not limited to, powder, tablets, pills,capsules and lozenges.

The dosage and frequency (single or multiple doses) administered to amammal can vary depending upon a variety of factors, for example,whether the mammal suffers from another disease, and its route ofadministration; size, age, sex, health, body weight, body mass index,and diet of the recipient; nature and extent of symptoms of the diseasebeing treated (e.g. symptoms of cancer and severity of such symptoms),kind of concurrent treatment, complications from the disease beingtreated or other health-related problems. Other therapeutic regimens oragents can be used in conjunction with the methods and compounds of theinvention. Adjustment and manipulation of established dosages (e.g.,frequency and duration) are well within the ability of those skilled inthe art.

For any composition (e.g., the conjugates provided herein includingembodiments thereof) described herein, the therapeutically effectiveamount can be initially determined from cell culture assays. Targetconcentrations will be those concentrations of active compound(s) thatare capable of achieving the methods described herein, as measured usingthe methods described herein or known in the art. As is well known inthe art, effective amounts for use in humans can also be determined fromanimal models. For example, a dose for humans can be formulated toachieve a concentration that has been found to be effective in animals.The dosage in humans can be adjusted by monitoring effectiveness andadjusting the dosage upwards or downwards, as described above. Adjustingthe dose to achieve maximal efficacy in humans based on the methodsdescribed above and other methods is well within the capabilities of theordinarily skilled artisan.

Dosages may be varied depending upon the requirements of the patient andthe compound being employed. The dose administered to a patient, in thecontext of the present invention should be sufficient to affect abeneficial therapeutic response in the patient over time. The size ofthe dose also will be determined by the existence, nature, and extent ofany adverse side-effects. Determination of the proper dosage for aparticular situation is within the skill of the practitioner. Generally,treatment is initiated with smaller dosages which are less than theoptimum dose of the compound. Thereafter, the dosage is increased bysmall increments until the optimum effect under circumstances isreached.

Dosage amounts and intervals can be adjusted individually to providelevels of the administered compound effective for the particularclinical indication being treated. This will provide a therapeuticregimen that is commensurate with the severity of the individual'sdisease state.

Utilizing the teachings provided herein, an effective prophylactic ortherapeutic treatment regimen can be planned that does not causesubstantial toxicity and yet is effective to treat the clinical symptomsdemonstrated by the particular patient. This planning should involve thecareful choice of active compound by considering factors such ascompound potency, relative bioavailability, patient body weight,presence and severity of adverse side effects, preferred

“Pharmaceutically acceptable excipient” and “pharmaceutically acceptablecarrier” refer to a substance that aids the administration of an activeagent to and absorption by a subject and can be included in thecompositions of the present invention without causing a significantadverse toxicological effect on the patient. Non-limiting examples ofpharmaceutically acceptable excipients include water, NaCl, normalsaline solutions, lactated Ringer's, normal sucrose, normal glucose,binders, fillers, disintegrants, lubricants, coatings, sweeteners,flavors, salt solutions (such as Ringer's solution), alcohols, oils,gelatins, carbohydrates such as lactose, amylose or starch, fatty acidesters, hydroxymethylcellulose, polyvinyl pyrrolidine, and colors, andthe like. Such preparations can be sterilized and, if desired, mixedwith auxiliary agents such as lubricants, preservatives, stabilizers,wetting agents, emulsifiers, salts for influencing osmotic pressure,buffers, coloring, and/or aromatic substances and the like that do notdeleteriously react with the compounds of the invention. One of skill inthe art will recognize that other pharmaceutical excipients are usefulin the present invention.

The term “pharmaceutically acceptable salt” refers to salts derived froma variety of organic and inorganic counter ions well known in the artand include, by way of example only, sodium, potassium, calcium,magnesium, ammonium, tetraalkylammonium, and the like; and when themolecule contains a basic functionality, salts of organic or inorganicacids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate,maleate, oxalate and the like.

The term “preparation” is intended to include the formulation of theactive compound with encapsulating material as a carrier providing acapsule in which the active component with or without other carriers, issurrounded by a carrier, which is thus in association with it.Similarly, cachets and lozenges are included. Tablets, powders,capsules, pills, cachets, and lozenges can be used as solid dosage formssuitable for oral administration.

Methods of Treating Cancer

The conjugates as provided herein including embodiments thereof areuseful, inter alia, for the treatment of cancer. Thus, in an aspect, amethod for treating cancer in a subject in need thereof is provided. Themethod includes administering to the subject a therapeutically effectiveamount of a cell penetrating nucleic acid conjugate as described hereinincluding embodiments thereof, thereby treating the cancer in thesubject.

In embodiments, the cancer is breast cancer, prostate cancer, ovariancancer, brain cancer, pancreatic cancer, melanoma, colon cancer, gastriccancer, head-and-neck cancer, liver cancer, lung cancer, cervicalcancer, sarcoma, leukemia, lymphoma, multiple myeloma. In embodiments,the cancer is breast cancer. In embodiments, the cancer is prostatecancer. In embodiments, the cancer is ovarian cancer. In embodiments,the cancer is brain cancer. In embodiments, the cancer is pancreaticcancer. In embodiments, the cancer is melanoma. In embodiments, thecancer is colon cancer. In embodiments, the cancer is gastric cancer. Inembodiments, the cancer is head-and-neck cancer. In embodiments, thecancer is liver cancer. In embodiments, the cancer is lung cancer. Inembodiments, the cancer is cervical cancer. In embodiments, the canceris sarcoma. In embodiments, the cancer is leukemia. In embodiments, thecancer is lymphoma. In embodiments, the cancer is multiple myeloma. Inembodiments, the cancer is metastatic lung cancer.

In embodiments, the method includes decreasing in the subject anexpression level of BIRC5 or BclXL relative to a standard control. Inembodiments, the method includes decreasing in the subject an expressionlevel of BIRC5 and BclXL relative to a standard control. In embodiments,the method includes decreasing in the subject an expression level ofBIRC5 relative to a standard control. In embodiments, the methodincludes decreasing in the subject an expression level of BclXL relativeto a standard control. In embodiments, the standard control is anexpression level of BIRC5 or BclXL detected in the absence of a cellpenetrating nucleic acid conjugate as described herein includingembodiments thereof.

The term “BIRC5,” also known as “survivin”, as referred to hereinincludes any of the recombinant or naturally-occurring forms of the geneencoding baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5),homologs or variants thereof that maintain BIRC5 activity (e.g. withinat least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activitycompared to BIRC5). In some aspects, variants have at least 90%, 95%,96%, 97%, 98%, 99% or 100% amino acid sequence identity across the wholesequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring BIRC5polypeptide. In embodiments, the BIRC5 gene is substantially identicalto the nucleic acid identified by the ENSEMBLE reference numberENSG00000089685 or a variant having substantial identity thereto. Theexpression level of BRIC5 may be determined by detecting levels of BIRC5mRNA or protein using methods known in the art. In embodiments, theBIRC5 mRNA is the nucleic acid sequence as identified by the Ensembl ID:ENST00000301633.8, homolog or functional fragment thereof. Inembodiments, the BIRC5 protein is the amino acid sequence as identifiedby Uniprot reference number 015392, homolog or functional fragmentthereof.

The term “Bcl-xL,” also known as BCL2L1, as referred to herein includesany of the recombinant or naturally-occurring forms of the gene encodingB-cell lymphoma-extra large (BclXL), homologs or variants thereof thatmaintain Bcl-xL activity (e.g. within at least 50%, 80%, 90%, 95%, 96%,97%, 98%, 99% or 100% activity compared to Bcl-xL). In some aspects,variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acidsequence identity across the whole sequence or a portion of the sequence(e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to anaturally occurring Bcl-XL polypeptide. In embodiments, the BclXL geneis substantially identical to the nucleic acid identified by theENSEMBLE reference number ENSG00000171552 or a variant havingsubstantial identity thereto. The expression level of BclXL may bedetermined by detecting levels of BclXL mRNA or protein using methodsknown in the art. In embodiments, the BclXL mRNA is the nucleic acidsequence as identified by the Ensembl ID: ENST00000307677.4, homolog orfunctional fragment thereof. In embodiments, the BIRC5 protein is theamino acid sequence as identified by Uniprot reference number Q07817,homolog or functional fragment thereof.

Methods of Increasing P53 in a Cell

The conjugates provided herein including embodiments thereof are furthercontemplated as a means of increasing p53 in a cell. A “p53 protein” or“p53” as referred to herein includes any of the recombinant ornaturally-occurring forms of the tumor protein p53 (p53) or variants orhomologs thereof that maintain p53 activity (e.g. within at least 50%,80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared top53). Insome aspects, the variants or homologs have at least 90%, 95%, 96%, 97%,98%, 99% or 100% amino acid sequence identity across the whole sequenceor a portion of the sequence (e.g. a 50, 100, 150 or 200 continuousamino acid portion) compared to a naturally occurring p53 protein. Inembodiments, the p53 protein is substantially identical to the proteinidentified by the UniProt reference number P04637 or a variant orhomolog having substantial identity thereto.

In an aspect, a method of increasing expression of p53 in a cancer cellis provided, the method including contacting a cancer cell with aneffective amount of a cell penetrating nucleic acid conjugate asdescribed herein including embodiments thereof, thereby increasingexpression of p53 in the cancer cell.

Methods of Inhibiting Tumor Vascularization

The conjugates provided herein including embodiments thereof are usefulfor the treatment of cancer through inhibition of tumor vascularization.Thus, in another aspect, a method of inhibiting tumor vascularization ina subject in need thereof is provided, the method includingadministering to the subject a therapeutically effective amount of acell penetrating nucleic acid conjugate as described herein includingembodiments thereof, thereby inhibiting tumor vascularization in thesubject.

Methods of Treating an Inflammatory Disease

The conjugates provided herein including embodiments thereof are alsouseful for the treatment of inflammatory disease. Therefore, in anotheraspect, a method of treating an inflammatory disease in a subject inneed thereof is provided, the method including administering to thesubject a therapeutically effective amount of a cell penetrating nucleicacid conjugate as described herein including embodiments thereof,thereby treating an inflammatory disease in the subject.

As used herein, the term “inflammatory disease” refers to a disease orcondition characterized by aberrant inflammation (e.g., an increasedlevel of inflammation compared to a control such as a healthy person notsuffering from a disease). Examples of inflammatory diseases includetraumatic brain injury, arthritis, rheumatoid arthritis, psoriaticarthritis, juvenile idiopathic arthritis, multiple sclerosis, systemiclupus erythematosus (SLE), myasthenia gravis, juvenile onset diabetes,diabetes mellitus type 1, Guillain-Barre syndrome, Hashimoto'sencephalitis, Hashimoto's thyroiditis, ankylosing spondylitis,psoriasis, Sjogren's syndrome, vasculitis, glomerulonephritis,auto-immune thyroiditis, Behcet's disease, Crohn's disease, ulcerativecolitis, bullous pemphigoid, sarcoidosis, ichthyosis, Gravesophthalmopathy, inflammatory bowel disease, Addison's disease, Vitiligo,asthma, allergic asthma, acne vulgaris, celiac disease, chronicprostatitis, inflammatory bowel disease, pelvic inflammatory disease,reperfusion injury, sarcoidosis, transplant rejection, interstitialcystitis, atherosclerosis, and atopic dermatitis.

In embodiments, the method includes decreasing in the subject anexpression level of FGA, IL1B or SERPINA3 relative to a standardcontrol. In embodiments, the method includes decreasing in the subjectan expression level of FGA, IL1B and SERPINA3 relative to a standardcontrol. In embodiments, the method includes decreasing in the subjectan expression level of FGA relative to a standard control. Inembodiments, the method includes decreasing in the subject an expressionlevel of IL1B relative to a standard control. In embodiments, the methodincludes decreasing in the subject an expression level of SERPINA3relative to a standard control. In embodiments, the standard control isan expression level of FGA, IL1B or SERPINA3 detected in the absence ofa cell penetrating nucleic acid conjugate as described herein includingembodiments thereof.

The term “FGA” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding fibrinogen alpha chain(FGA), homologs or variants thereof that maintain FGA activity (e.g.within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activitycompared to FGA). In some aspects, variants have at least 90%, 95%, 96%,97%, 98%, 99% or 100% amino acid sequence identity across the wholesequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring FGApolypeptide. In embodiments, the FGA gene is substantially identical tothe nucleic acid identified by the ENSEMBLE reference numberENSG00000171560 or a variant having substantial identity thereto. Theexpression level of FGA may be determined by detecting levels of FGAmRNA or protein using methods known in the art. In embodiments, the FGAmRNA is the nucleic acid sequence as identified by the Ensembl ID:ENST00000302053.7, homolog or functional fragment thereof. Inembodiments, the FGA protein is the amino acid sequence as identified byUniprot reference number P02671, homolog or functional fragment thereof.

The term “IL1B” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding interleukin 1 beta(IL1B), homologs or variants thereof that maintain IL1B activity (e.g.within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activitycompared to IL1B). In some aspects, variants have at least 90%, 95%,96%, 97%, 98%, 99% or 100% amino acid sequence identity across the wholesequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring IL1Bpolypeptide. In embodiments, the IL1B gene is substantially identical tothe nucleic acid identified by the ENSEMBLE reference numberENSG00000125538 or a variant having substantial identity thereto. Theexpression level of IL1B may be determined by detecting levels of IL1BmRNA or protein using methods known in the art. In embodiments, the IL1BmRNA is the nucleic acid sequence as identified by the Ensembl ID:ENST00000263341.6, homolog or functional fragment thereof. Inembodiments, the IL1B protein is the amino acid sequence as identifiedby Uniprot reference number P01584, homolog or functional fragmentthereof.

The term “SERPINA3” as referred to herein includes any of therecombinant or naturally-occurring forms of the gene encoding alpha1-antichymotrypsin, homologs or variants thereof that maintain alpha1-antichymotrypsin activity (e.g. within at least 50%, 80%, 90%, 95%,96%, 97%, 98%, 99% or 100% activity compared to alpha1-antichymotrypsin). In some aspects, variants have at least 90%, 95%,96%, 97%, 98%, 99% or 100% amino acid sequence identity across the wholesequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring alpha1-antichymotrypsin polypeptide. In embodiments, the SERPINA3 gene issubstantially identical to the nucleic acid identified by the ENSEMBLEreference number ENSG00000196136 or a variant having substantialidentity thereto. The expression level of SERPINA3 may be determined bydetecting levels of SERPINA3 mRNA or protein using methods known in theart. In embodiments, the SERPINA mRNA is the nucleic acid sequence asidentified by the Ensembl ID: ENST00000467132.5, homolog or functionalfragment thereof. In embodiments, the alpha 1-antichymotrypsin proteinis the amino acid sequence as identified by Uniprot reference numberP01011, homolog or functional fragment thereof.

Non-Opioid-Based Methods of Treating Pain

Opioids are a class of compound among the most widely used for treatmentof pain. Opioid drugs produce effects by interacting with opioidreceptors. Opioids have opium- or morphine-like properties allowing themto act as opioid receptor agonists. However, opioids have otherpharmacological effects including drowsiness, respiratory depression,and constipation, as well as abuse potential and tolerance. The negativeside-effects of opioid use have spurred a need for non-opioid-based paintreatments. Provided herein are, inter alia, non-opioid-based methodsfor treating pain in a subject in need thereof.

In an aspect, a method of treating pain in a subject in need thereof isprovided. The method includes administering to the subject atherapeutically effective amount of a cell penetrating nucleic acidconjugate as described herein including embodiments thereof, therebytreating pain in the subject.

The pain may emanate from a wide variety of sources or be derived from awide variety of causes. Thus, the pain may be nociceptive pain (e.g.,trauma, procedural, cut, sprains, bone fractures, burns, bumps,bruises), neuropathic pain (e.g., post herpetic neuralgia, reflexsympathetic dystrophy/causalgia, cancer pain, pain induced by treatmentof cancer, HIV/AIDS or hepatitis, diabetes, phantom limb pain,entrapment neuropathy, chronic alcohol use, exposure to other toxins,vitamin deficiencies and idiopathic), inflammatory pain (e.g.,arthritis, colitis, carditis, pulmonits, nephritis, myositis,vasculitis, endometriosis, neuritis, dermatitis and pain associated withother inflammatory conditions), chronic widespread pain (e.g.,fibromyalgia, migraine, irritable bowel syndrome, syndrome X,interstitial bladder syndrome, chronic fatigue syndrome, post-traumaticstress disorder, pain associated with psychiatric illnesses such asanxiety and depression and stress-related pain conditions, and secondaryto inflammatory or neuropathic pain syndromes) or mixed etiology (i.e.,combinations of two or more of the above four categories).

In embodiments, the nucleic acid conjugates useful for treating pain inthe methods provided herein does not mediate its analgesic effectthrough opioid receptors. In embodiments, the nucleic acid conjugateuseful for treating pain in the methods provided herein does not haveopium- or morphine-like properties. In embodiments, the nucleic acidconjugate useful for treating pain in the methods provided herein is notan opioid receptor ligand. In embodiments, the nucleic acid conjugateuseful for treating pain in the methods provided herein does not bind toan opioid receptor. In embodiments, the nucleic acid conjugate usefulfor treating pain in the methods provided herein is not an opioidreceptor agonist.

In embodiments, the method includes decreasing in the subject anexpression level of PTGS1, PTGS2, CALCA or SST relative to a standardcontrol. In embodiments, the method includes decreasing in the subjectan expression level of PTGS1, PTGS2, CALCA and SST relative to astandard control. In embodiments, the method includes decreasing in thesubject an expression level of PTG51 relative to a standard control. Inembodiments, the method includes decreasing in the subject an expressionlevel of PTG52 relative to a standard control. In embodiments, themethod includes decreasing in the subject an expression level of CALCArelative to a standard control. In embodiments, the method includesdecreasing in the subject an expression level of SST relative to astandard control. In embodiments, the standard control is an expressionlevel of PTGS1, PTGS2, CALCA or SST detected in the absence of a cellpenetrating nucleic acid conjugate as described herein includingembodiments thereof.

The term “PTGS1” as referred to herein includes any of the recombinantor naturally-occurring forms of the gene encodingprostaglandin-endoperoxide synthase 1 (PTGS1), also known as COX-1,homologs or variants thereof that maintain PTGS1 activity (e.g. withinat least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activitycompared to PTGS1). In some aspects, variants have at least 90%, 95%,96%, 97%, 98%, 99% or 100% amino acid sequence identity across the wholesequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring PTGS1polypeptide. In embodiments, the PTGS1 gene is substantially identicalto the nucleic acid identified by the ENSEMBLE reference numberENSG00000095303 or a variant having substantial identity thereto. Theexpression level of PTGS1 may be determined by detecting levels of PTGS1mRNA or protein using methods known in the art. In embodiments, thePTGS1 mRNA is the nucleic acid sequence as identified by the Ensembl ID:ENST00000540753.5, homolog or functional fragment thereof. Inembodiments, the PTGS1 protein is the amino acid sequence as identifiedby Uniprot reference number P23219, homolog or functional fragmentthereof.

The term “PTGS2” as referred to herein includes any of the recombinantor naturally-occurring forms of the gene encodingprostaglandin-endoperoxide synthase 2 (PTGS2), also known as COX-2,homologs or variants thereof that maintain PTGS2 activity (e.g. withinat least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activitycompared to PTGS2). In some aspects, variants have at least 90%, 95%,96%, 97%, 98%, 99% or 100% amino acid sequence identity across the wholesequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring PTGS2polypeptide. In embodiments, the PTGS2 gene is substantially identicalto the nucleic acid identified by the ENSEMBLE reference numberENSG00000073756 or a variant having substantial identity thereto. Theexpression level of PTGS2 may be determined by detecting levels of PTGS2mRNA or protein using methods known in the art. In embodiments, thePTGS2 mRNA is the nucleic acid sequence as identified by the Ensembl ID:ENST00000367468.9, homolog or functional fragment thereof. Inembodiments, the PTGS2 protein is the amino acid sequence as identifiedby Uniprot reference number P35354, homolog or functional fragmentthereof.

The term “SST” as referred to herein includes any of the recombinant ornaturally-occurring forms of the gene encoding somatostatin (SST),homologs or variants thereof that maintain SST activity (e.g. within atleast 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity comparedto SST). In some aspects, variants have at least 90%, 95%, 96%, 97%,98%, 99% or 100% amino acid sequence identity across the whole sequenceor a portion of the sequence (e.g. a 50, 100, 150 or 200 continuousamino acid portion) compared to a naturally occurring SST polypeptide.In embodiments, the SST gene is substantially identical to the nucleicacid identified by the ENSEMBLE reference number ENSG00000157005 or avariant having substantial identity thereto. The expression level of SSTmay be determined by detecting levels of SST mRNA or protein usingmethods known in the art. In embodiments, the SST mRNA is the nucleicacid sequence as identified by the Ensembl ID: ENST00000287641.3,homolog or functional fragment thereof. In embodiments, the SST proteinis the amino acid sequence as identified by Uniprot reference numberP61278, homolog or functional fragment thereof.

The term “CALCA” as referred to herein includes any of the recombinantor naturally-occurring forms of the gene encoding calcitoningene-related peptide (CGRP), homologs or variants thereof that maintainCALCA activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%,99% or 100% activity compared to CALCA). In some aspects, variants haveat least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequenceidentity across the whole sequence or a portion of the sequence (e.g. a50, 100, 150 or 200 continuous amino acid portion) compared to anaturally occurring CALCA polypeptide. In embodiments, the CALCA gene issubstantially identical to the nucleic acid identified by the ENSEMBLEreference number ENSG00000110680 or a variant having substantialidentity thereto. The expression level of CALCA may be determined bydetecting levels of CALCA mRNA or protein using methods known in theart. In embodiments, the CALCA mRNA is the nucleic acid sequence asidentified by the Ensembl ID: ENST00000361010.7, homolog or functionalfragment thereof. In embodiments, the CALCA protein is the amino acidsequence as identified by Uniprot reference numbers P01258, P06881,homolog or functional fragment thereof.

Methods of Decreasing IL-6 Signaling in a Cell

The conjugates provided herein including embodiments thereof are usefulfor decreasing IL-6 signaling in a cell. Thus, in an aspect, a method ofinhibiting IL-6 signaling in a cell is provided, the method includingcontacting a cell with an effective amount of a cell penetrating nucleicacid conjugate as provided herein including embodiments thereof, therebyinhibiting IL-6 signaling in the cell.

Methods of Delivering a Non-Cell Penetrating Nucleic Acid into a Cell

The conjugates provided herein including embodiments thereof are usefulfor delivering non-cell penetrating nucleic acids into a cell. In anaspect is provided a method of delivering a non-cell penetrating nucleicacid into a cell, the method including contacting a cell with the cellpenetrating nucleic acid conjugate as provided herein includingembodiments thereof, thereby delivering the non-cell penetrating nucleicacid into the cell.

EXAMPLES

Here, Applicants describe a gymnotic administration of miRNAs, forexample, let7a-3p (SEQ ID NO:1), let7a-5p (SEQ ID NO:2), miR17-3p (SEQID NO:3), miR17-5p (SEQ ID NO:4), miR218-5p (SEQ ID NO:5), by protectingthe operating miRNA sequences through extending the miRNAs on their 3′ends with phosphorothioated ssDNA oligonucleotides or phosphorothioatedsingle-stranded abasic sugar-phosphate backbone polymers. Applicantsshow that attachment of phosphorothioated ssDNA oligonucleotides orphosphorothioated single-stranded abasic sugar-phosphate backbonepolymers to miRNAs on the 3′ ends of the miRNAs facilitatesintracellular delivery of miRNAs into tumor cells and subsequent targetof their target genes as well as protection of miRNAs from enzymaticdegradation in serum.

Example 1: Production of Cell Internalizing Nucleic Acid Compounds ViaCovalent Linkage to Phosphorothioated SSDNA Oligonucleotides

miRNAs with naturally occurring sequences were fused covalently tophosphorothioated ssDNA (PS) 20meric oligo to facilitate cellularinternalization targeting intracellular molecular targets. Anon-phosphorothioated, phosphodiester ssDNA oligo (PO) extension of themiRNAs was employed as a non-internalizing control.

Applicants modified naturally occurring miRNAs, for example, let7a-3p(SEQ ID NO:1) (FIG. 1 ), let7a-5p (SEQ ID NO:2) (FIG. 3 ), miR17-3p (SEQID NO:3) (FIG. 5 ), miR17-5p (SEQ ID NO:4) (FIG. 7 ), and miR218-5p (SEQID NO:5) (FIG. 9 ) by attaching a phosphorothioated ssDNA (PS) 20mericoligo to the 3′ end of the miRNAs via a chemical linker. Examples of aphosphorothioated ssDNA (PS) 20meric oligo include, but are not limitedto, SEQ ID NO:6 (TCCATGAGCTTCCTGATGCT) and SEQ ID NO:7(AGCATCAGGAAGCTCATGGA). Applicants designed that the modification byssDNA oligo avoids any C/G or G/C motifs, because it is known that CpGoligodeoxynucleotides (CpG-ODN) involve undesired Toll-like receptor(TLR) engagement and subsequent intracellular signaling. Applicants usedan alkyl chain harboring a fluorophore as a linker to track theconjugate molecule.

Example 2: Modified MIRNA Elongated with Phosphorothioated SSDNAOligonucleotides Undergo Cellular Internalization

Once miRNAs were modified by elongation and fluorescently marked toenable intracellular tracking of modified miRNAs, Applicants assessedcellular internalization of PS-modified miRNAs by flow cytometryincluding PO-modified miRNA as negative non-internalizing controls.Human multiple myeloma cells MM.1 S were incubated either for 30 min orfor 48 hrs with modified miRNA as indicated and analyzed by flowcytometry to assess cellular load of cells with modified miRNA. Formodified let7a-3p miRNA (FIGS. 2A and 2B) and modified let7a-5p miRNA(FIGS. 4A and 4B), 10 μg/ml was used for both 30 min and 48 hrincubation. For miR17-3p miRNA (FIGS. 6A and 6B), modified miR17-5pmiRNA (FIGS. 8A and 8B) and modified miR218-5p miRNA (FIGS. 10A and10B), 20 μg/ml was used for 30 min incubation and 10 μg/ml was used for48 hr incubation, respectively.

Example 3: Modified MIRNA Elongated with Phosphorothioated SSDNAOligonucleotides Reducing STAT3 Target Gene Expression

Human multiple myeloma cancer cells are known to undergo increased celldivision through IL-6-triggered STAT3 signaling. Numerous studies haveshown that let7a-3p miRNA (SEQ ID NO:1), let7a-5p miRNA (SEQ ID NO:2),miR17-3p miRNA (SEQ ID NO:3), miR17-5p miRNA (SEQ ID NO:4), or miR218-5pmiRNA (SEQ ID NO:5) inhibits the activity of transcription factor SignalTransducer and Activator of Transcription 3 (STAT3). Human multiplemyeloma cells MM.1S were incubated for 48 hrs daily with 10 μg/mlmodified miRNA as indicated and expression of the STAT3 target genes wasanalyzed by RT-PCR. As shown in FIGS. 2C, 4C, 6C, 8C and 10C, incubationwith PS-modified let7a-3p miRNA (SEQ ID NO:1), let7a-5p miRNA (SEQ IDNO:2), miR17-3p miRNA (SEQ ID NO:3), miR17-5p miRNA (SEQ ID NO:4), ormiR218-5p miRNA (SEQ ID NO:5) inhibited expression of STAT3 targetgenes, for example, oncogenic Bcl-xL and/or IL-6 genes.

Example 4: Production of Cell Internalizing Nucleic Acid Compounds ViaCovalent Linkage to Phosphorothioated Single-Stranded AbasicSugar-Phosphate Backbone Polymers

miRNAs with naturally occurring sequences were fused covalently to aphosphorothioated single-stranded abasic sugar-phosphate backbone (PS)20meric polymer to facilitate cellular internalization targetingintracellular molecular targets. A non-phosphorothioated, phosphodiestersingle-stranded abasic sugar-phosphate backbone polymer (PO) extensionof the miRNAs was employed as a non-internalizing control.

Applicants modified naturally occurring miRNAs, for example, let7a-5p(SEQ ID NO:2) by attaching a phosphorothioated single-stranded abasicsugar-phosphate backbone (PS) 20meric polymer to the 3′ end of themiRNAs via a chemical linker (FIG. 11A). Applicants designed themodification based on that phosphorothioated ssDNA oligo enablessuccessful intracellular delivery, but bases (nucleic acids) may not berequired to facilitate intracellular delivery of the conjugate and thusmay be excluded. FIG. 11B schematically shows the abasic sugar-phosphatemodule (referred as “D”) lacking a base (a nucleic acid) in comparisonto basic “spacers.” Applicants used an alkyl chain harboring afluorophore as a linker to track the conjugate molecule.

Example 5: Modified MIRNA Elongated with PhosphorothioatedSingle-Stranded Abasic Sugar-Phosphate Backbone Polymers UndergoCellular Internalization

Once miRNAs were modified by elongation and fluorescently marked toenable intracellular tracking of modified miRNAs, Applicants assessedcellular internalization of PS polymer-modified miRNAs by flow cytometryincluding PO polymer-modified miRNA as negative non-internalizingcontrols. Human multiple myeloma cells MM.1S were incubated for 30 min(FIG. 12A) with 20 μg/ml polymer-modified let7a-5p miRNA as indicatedand analyzed by flow cytometry to assess cellular load of cells withmodified miRNA.

Example 6: Modified MIRNA Elongated with PhosphorothioatedSingle-Stranded Abasic Sugar-Phosphate Backbone Polymers Reducing Stat3Target Gene Expression

Human multiple myeloma cancer cells are known to undergo increased celldivision through IL-6-triggered STAT3 signaling. Numerous studies haveshown that let7a-5p miRNA (SEQ ID NO:2) inhibits the activity of SignalTransducer and Activator of Transcription 3 (STAT3). Human multiplemyeloma cells MM.1S were incubated for 48 hrs daily with 10 μg/mlpolymer-modified let7a-5p miRNA as indicated and expression of the STAT3target gene, oncogenic Bcl-xL gene, was analyzed by RT-PCR. As shown inFIG. 12B, incubation with PS polymer-modified let7a-5p miRNA inhibitedexpression of Bcl-xL gene.

REFERENCES

-   Herrmann, A., Nachaev, S., Lahtz, C., Armstrong, B., Kowolik, C.,    Kortylewski, M., Jove, R., and Hua, Y. (2014). STAT3 nuclear egress    requires exportin 7 via engaging lysine acetylation. MOJ Cell Sci    Report 1(1): 00004. DOI: 10.15406/mojcsr.2014.01.00004.

P EMBODIMENTS

P Embodiment 1. A cell penetrating nucleic acid conjugate comprising:

-   -   (i) a non-cell penetrating ribonucleic acid compound comprising        the sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID        NO:4 or SEQ ID NO:5;    -   (ii) a phosphorothioate polymer; and    -   (iii) a chemical linker attaching said phosphorothioate polymer        to the 3′ end of said non-cell penetrating ribonucleic acid        compound; wherein said phosphorothioate polymer enhances        intracellular delivery of the non-cell penetrating nucleic acid        compound.

P Embodiment 2. The conjugate of P Embodiment 1, wherein said non-cellpenetrating ribonucleic acid compound is a micro RNA (miRNA).

P Embodiment 3. The conjugate of P Embodiment 1 or 2, wherein saidnon-cell penetrating ribonucleic acid compound is about 10, 20, 30, 40,50, 60, 70, 80, 90 or more residues in length.

P Embodiment 4. The conjugate of any one of P Embodiments 1-3, whereinsaid non-cell penetrating ribonucleic acid compound is from about 20 toabout 30 residues in length.

P Embodiment 5. The conjugate of any one of P Embodiments 1-4, whereinsaid non-cell penetrating ribonucleic acid compound is from about 20 toabout 25 residues in length.

P Embodiment 6. The conjugate of any one of P Embodiments 1-5, whereinsaid non-cell penetrating ribonucleic acid compound is 21, 22 or 23residues in length.

P Embodiment 7. The conjugate of any one of P Embodiments 1-6, whereinsaid phosphorothioate polymer is a phosphorothioate nucleic acid or anabasic sugar-phosphorothioated polymer.

P Embodiment 8. The conjugate of any one of P Embodiments 1-7, whereinsaid phosphorothioate polymer is a phosphorothioate deoxyribonucleicacid.

P Embodiment 9. The conjugate of any one of P Embodiments 1-8, whereinsaid phosphorothioate polymer is about 10, 20, 30, 40, 50, 60, 70, 80,90, 100 or more residues in length.

P Embodiment 10. The conjugate of any one of P Embodiments 1-9, whereinsaid phosphorothioate polymer is from about 10 to about 30 residues inlength.

P Embodiment 11. The conjugate of any one of P Embodiments 1-10, whereinsaid phosphorothioate polymer is about 20 residues in length.

P Embodiment 12. The conjugate of any one of P Embodiments 1-11, whereinsaid phosphorothioate polymer comprises the sequence of SEQ ID NO:6 orSEQ ID NO:7.

P Embodiment 13. The conjugate of any one of P Embodiments 1-12, whereinsaid phosphorothioate polymer is single-stranded.

P Embodiment 14. The conjugate of any one of P Embodiments 1-13, whereinsaid chemical linker is a covalent linker.

P Embodiment 15. The conjugate of any one of P Embodiments 1-14, whereinsaid chemical linker is a non-immunogenic linker.

P Embodiment 16. The conjugate of any one of P Embodiments 1-15, whereinsaid conjugate further comprises a detectable moiety.

P Embodiment 17. The conjugate of P Embodiment 16, wherein saiddetectable moiety is attached to said non-cell penetrating ribonucleicacid compound.

P Embodiment 18. The conjugate of P Embodiment 16, wherein saiddetectable moiety is attached to said phosphorothioate polymer.

P Embodiment 19. The conjugate of any one of P Embodiments 1-18, whereinsaid non-cell penetrating ribonucleic acid compound inhibits STAT3activity relative to a standard control.

P Embodiment 20. The conjugate of any one of P Embodiments 1-19, whereinsaid non-cell penetrating ribonucleic acid compound inhibits expressionof a STAT3 target gene relative to a standard control.

P Embodiment 21. The conjugate of P Embodiment 20, wherein said STAT3target gene is an oncogene.

P Embodiment 22. The conjugate of P Embodiment 20, wherein said STAT3target gene is Bcl-xL or IL-6.

P Embodiment 23. A cell comprising a cell penetrating nucleic acidconjugate of any one of P Embodiments 1-22.

P Embodiment 24. A pharmaceutical composition comprising a cellpenetrating nucleic acid conjugate of any one of P Embodiments 1-22 anda pharmaceutically acceptable carrier.

P Embodiment 25. A method of treating cancer in a subject in needthereof, said method comprising administering to said subject atherapeutically effective amount of a cell penetrating nucleic acidconjugate of any one of P Embodiments 1-22, thereby treating said cancerin said subject.

P Embodiment 26. The method of P Embodiment 25, wherein said cancer isbreast cancer, prostate cancer, ovarian cancer, brain cancer, pancreaticcancer, melanoma, colon cancer, gastric cancer, head-and-neck cancer,liver cancer, lung cancer, cervical cancer, sarcoma, leukemia, lymphoma,multiple myeloma.

P Embodiment 27. The method of P Embodiment 26, said method comprisingdecreasing in said subject an expression level of BIRC5 or Bcl-xLrelative to a standard control.

P Embodiment 28. A method of increasing expression of p53 in a cancercell, said method comprising contacting a cancer cell with an effectiveamount of a cell penetrating nucleic acid conjugate of any one of PEmbodiments 1-22, thereby increasing expression of p53 in said cancercell.

P Embodiment 29. A method of inhibiting tumor vascularization in asubject in need thereof, said method comprising administering to saidsubject a therapeutically effective amount of a cell penetrating nucleicacid conjugate of any one of P Embodiments 1-22, thereby inhibitingtumor vascularization in said subject.

P Embodiment 30. A method of treating an inflammatory disease in asubject in need thereof, said method comprising administering to saidsubject a therapeutically effective amount of a cell penetrating nucleicacid conjugate of any one of P Embodiments 1-22, thereby treating aninflammatory disease in said subject.

P Embodiment 31. The method of P Embodiment 30, said method comprisingdecreasing in said subject an expression level of FGA, IL1B or SERPINA3relative to a standard control.

P Embodiment 32. A method of treating pain in a subject in need thereof,said method comprising administering to said subject a therapeuticallyeffective amount of a cell penetrating nucleic acid conjugate of any oneof P Embodiments 1-22, thereby treating pain in said subject.

P Embodiment 33. The method of P Embodiment 32, said method comprisingdecreasing in said subject an expression level of PTGS1, PTGS2, CALCA orSST relative to a standard control.

P Embodiment 34. A method of inhibiting IL-6 signaling in a cell, saidmethod comprising contacting a cell with an effective amount of a cellpenetrating nucleic acid conjugate of any one of P Embodiments 1-22,thereby inhibiting IL-6 signaling in said cell.

P Embodiment 35. A method of delivering a non-cell penetrating nucleicacid into a cell, said method comprising contacting a cell with a cellpenetrating nucleic acid conjugate of any one of P Embodiments 1-22,thereby delivering said non-cell penetrating nucleic acid into saidcell.

INFORMAL SEQUENCE LISTING let7a-3p miRNA (SEQ ID NO: 1):CUAUACAAUCUACUGUCUUUC let7a-5p miRNA (SEQ ID NO: 2):UGAGGUAGUAGGUUGUAUAGUU miR17-3p miRNA (SEQ ID NO: 3):ACUGCAGUGAAGGCACUUGUAG miR17-5p miRNA (SEQ ID NO: 4):CAAAGUGCUUACAGUGCAGGUAG miR218-5p miRNA (SEQ ID NO: 5):UUGUGCUUGAUCUAACCAUGU phosphorothioate polymer (SEQ ID NO: 6):TCCATGAGCTTCCTGATGCT phosphorothioate polymer (SEQ ID NO: 7):AGCATCAGGAAGCTCATGGA STAT3 polypeptide (SEQ ID NO: 8):MAQWNQLQQLDTRYLEQLHQLYSDSFPMELRQFLAPWIESQDWAYAASKESHATLVFHNLLGEIDQQYSRFLQESNVLYQHNLRRIKQFLQSRYLEKPMEIARIVARCLWEESRLLQTAATAAQQGGQANHPTAAVVTEKQQMLEQHLQDVRKRVQDLEQKMKVVENLQDDFDFNYKTLKSQGDMQDLNGNNQSVTRQKMQQLEQMLTALDQMRRSIVSELAGLLSAMEYVQKTLTDEELADWKRRQQIACIGGPPNICLDRLENWITSLAESQLQTRQQIKKLEELQQKVSYKGDPIVQHRPMLEERIVELFRNLMKSAFVVERQPCMPMHPDRPLVIKTGVQFTTKVRLLVKFPELNYQLKIKVCIDKDSGDVAALRGSRKFNILGTNTKVMNMEESNNGSLSAEFKHLTLREQRCGNGGRANCDASLIVTEELHLITFETEVYHQGLKIDLETHSLPVVVISNICQMPNAWASILWYNMLTNNPKNVNFFTKPPIGTWDQVAEVLSWQFSSTTKRGLSIEQLTTLAEKLLGPGVNYSGCQITWAKFCKENMAGKGFSFWVWLDNIIDLVKKYILALWNEGYIMGFISKERERAILSTKPPGTFLLRFSESSKEGGVTFTWVEKDISGKTQIQSVEPYTKQQLNNMSFAEIIMGYKIMDATNILVSPLVYLYPDIPKEEAFGKYCRPESQEHPEADPGSAAPYLKTKFICVTPTTCSNTIDLPMSPRTLDSLMQFGNNGEGAEPSAGGQFESLTFDMELTSEC ATSPM

What is claimed is:
 1. A method of treating multiple myeloma in asubject in need thereof, the method comprising administering to thesubject a therapeutically effective amount of a nucleic acid conjugatecomprising: (i) a microRNA selected from the group consisting oflet7a-3p miRNA, let7a-5p miRNA, miR17-3p miRNA, miR17-5p miRNA, andmiR218-5p miRNA; (ii) a phosphorothioate polymer selected from the groupconsisting of: (a) a single-stranded phosphorothioate deoxyribonucleicacid comprising from 15 to 30 nucleotide residues, and (b) asingle-stranded abasic sugar-phosphorothioated nucleic acid comprisingfrom 15 to 30 nucleotide residues; and (iii) a chemical linkercovalently attaching the phosphorothioate polymer to the 3′ end of themicroRNA; thereby treating the multiple myeloma in the subject.
 2. Amethod of inhibiting IL-6 signaling in a cell, the method comprisingcontacting a cell with an effective amount of a nucleic acid conjugatecomprising: (i) a microRNA selected from the group consisting oflet7a-3p miRNA, let7a-5p miRNA, miR17-3p miRNA, miR17-5p miRNA, andmiR218-5p miRNA; (ii) a phosphorothioate polymer selected from the groupconsisting of: (a) a single-stranded phosphorothioate deoxyribonucleicacid comprising from 15 to 30 nucleotide residues, and (b) asingle-stranded abasic sugar-phosphorothioated nucleic acid comprisingfrom 15 to 30 nucleotide residues; and (iii) a chemical linkercovalently attaching the phosphorothioate polymer to the 3′ end of themicroRNA; thereby inhibiting IL-6 signaling in the cell.
 3. A method ofdelivering a non-cell penetrating ribonucleic acid into a cell, themethod comprising contacting a cell with a cell penetrating ribonucleicacid conjugate wherein the cell penetrating ribonucleic acid conjugatecomprises: (i) a non-cell penetrating ribonucleic acid compound; (ii) aphosphorothioate nucleic acid; and (iii) a chemical linker attaching thephosphorothioate polymer to the non-cell penetrating ribonucleic acidcompound; thereby delivering the non-cell penetrating ribonucleic acidinto the cell.
 4. The method of claim 3, wherein the non-cellpenetrating ribonucleic acid compound is microRNA.
 5. The method ofclaim 4, wherein the microRNA is let7a-3p miRNA, let7a-5p miRNA,miR17-3p miRNA, miR17-5p miRNA, or miR218-5p miRNA.
 6. The method ofclaim 4, wherein the microRNA comprises SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, or SEQ ID NO:5.
 7. The method of claim 3, wherein thephosphorothioate polymer is a phosphorothioate deoxyribonucleic acid oran abasic sugar-phosphorothioated polymer.
 8. The method of claim 3,wherein the phosphorothioate polymer is: (a) a single-strandedphosphorothioate deoxyribonucleic acid comprising from 15 to 30nucleotide residues or (b) a single-stranded abasicsugar-phosphorothioated nucleic acid comprising from 15 to 30 nucleotideresidues.
 9. The method of claim 3, wherein the phosphorothioate polymercomprises SEQ ID NO: 6 or SEQ ID NO:
 7. 10. The method of claim 3,wherein the chemical linker attaches the phosphorothioate polymer to the3′ end of the non-cell penetrating ribonucleic acid compound.
 11. Themethod of claim 1, wherein the let7a-3p miRNA has SEQ ID NO:1, thelet7a-5p miRNA has SEQ ID NO:2, the miR17-3p miRNA has SEQ ID NO:3, themiR17-5p miRNA has SEQ ID NO:4, and the miR218-5p miRNA has SEQ ID NO:5.12. The method of claim 1, wherein the phosphorothioate polymer is thesingle-stranded phosphorothioate deoxyribonucleic acid comprising from15 to 30 nucleotide residues.
 13. The method of claim 12, wherein thesingle-stranded phosphorothioate deoxyribonucleic acid comprises from 18to 22 nucleotide residues.
 14. The method of claim 1, wherein thephosphorothioate polymer is the single-stranded abasicsugar-phosphorothioated nucleic acid comprising from 15 to 30 nucleotideresidues.
 15. The method of claim 14, wherein the single-stranded abasicsugar-phosphorothioated nucleic acid comprises from 18 to 22 nucleotideresidues.
 16. The method of claim 1, wherein the phosphorothioatepolymer comprises SEQ ID NO: 6 or SEQ ID NO:
 7. 17. The method of claim2, wherein the let7a-3p miRNA has SEQ ID NO:1, the let7a-5p miRNA hasSEQ ID NO:2, the miR17-3p miRNA has SEQ ID NO:3, the miR17-5p miRNA hasSEQ ID NO:4, and the miR218-5p miRNA has SEQ ID NO:5.
 18. The method ofclaim 2, wherein the phosphorothioate polymer is a single-strandedphosphorothioate deoxyribonucleic acid comprising from 18 to 22nucleotide residues.
 19. The method of claim 2, wherein thephosphorothioate polymer is a single-stranded abasicsugar-phosphorothioated nucleic acid comprising from 18 to 22 nucleotideresidues.
 20. The method of claim 2, wherein the phosphorothioatepolymer comprises SEQ ID NO: 6 or SEQ ID NO: 7.